3, 4-Methylenedioxy-substituted chalcones as therapeutic agents

ABSTRACT

The present invention pertains to the use of a compounds for the manufacture of a medicament for use in the treatment of a proliferative condition, wherein the compounds have the following formula:  
                 
 
     wherein: each of R B2 , R B3 , R B4 , and R B5  is independently —H, —OH, or —OMe; each of R 1  and R 2  is independently: —H, optionally substituted C 1-4 alkyl, or optionally substituted C 5-20 aryl; R A3  is —H, —OH, —OC(═O)R E , —OS(═O) 2 OH, or —OP(═O)(OH) 2 ; R E  is: —H, optionally substituted C 1-6 alkyl, optionally substituted C 3-20 heterocyclyl, or optionally substituted C 5-20 aryl; or a pharmaceutically acceptable salt, solvate, amide, ester, ether, chemically protected form, or prodrug thereof. The present invention also pertains to such compounds, pharmaceutical compositions comprising such compounds, and the use of such compounds and compositions, both in vitro and in vivo, for both diagnosis and treatment of, for example, proliferative conditions, such as cancer, and inflammatory conditions.

RELATED APPLICATION

[0001] This application is related to (and where permitted by law,claims priority to) United Kingdom patent application number GB0123777.5 filed 03 Oct. 2001, the contents of which are incorporatedherein by reference in their entirety.

TECHNICAL FIELD

[0002] This invention pertains to substituted chalcones, specificallysubstituted 1-(3,4-methylenedioxy)-3-phenyl-prop-1-en-3-ones, which havetherapeutic application, for example, as potent antiproliferative agentsand antiinflammatory agents. The present invention also pertains topharmaceutical compositions comprising such compounds, and the use ofsuch compounds and compositions, both in vitro and in vivo, for bothdiagnosis and treatment of, for example, proliferative conditions, suchas cancer, and inflammatory conditions.

BACKGROUND

[0003] Many clinically successful anticancer drugs are themselves eithernatural products or have been developed from naturally occurring leadcompounds. Great interest is currently being paid to drugs isolated fromnatural resources which have already been used as a medicine. The driedwhole plant of Scutellaria barbata D. Don (Labiatae) is used inTraditional Chinese Medicine as an anti-inflammatory, an antitumouragent, and a diuretic. The α,β-unsaturated ketone,(E)-1-(4′-hydroxyphenyl)but-1-en-3-one has been isolated from this plantand found to have moderate antitumour activity (IC50 of 60 μM for K562).

[0004] Various analogues of this compounds have been examined forantitumour activity, including one class of analogs, chalcones.

[0005] Chalcone, also known as chalkone, benzylideneacetophenone,benzalacetophenone, and phenyl styryl ketone, is1,3-diphenyl-2-propen-1-one, and has the following structure:

[0006] A number of substituted chalcones have been prepared, with one ormore substituents on the styryl phenyl group (left, A), the acyl phenylgroup (right, B), and/or the double bond carbon atoms.

[0007] A number of substituted chalcones with apparent biologicalactivity have been reported.

[0008] Hall et al., 1981, describe a number of substituted chalconeswhich were alleged to have anti-inflammatory properties. The recitedcompounds are shown below (see Example 10, therein) (substituent is Hunless otherwise specified): 1 (X═OH, Z=OH, L=OH), 2 (X═OH, Y═OH, Z=OH,L=OMe), 3 (Y═OH, L=NMe₂), 4 (Y═OH, L=Cl), 5 (Y═OH, K=OEt, L=OH), 6(Y═OH, K=C₆H₅F), 7 (Y═OH, L=OH), 8 (Y═OMe, K=OMe), 9 (Y═OH, J=F), and 10(Y═OMe, L=OH).

[0009] Eda Shoei et al., 1986, describe several substituted chalconeswhich were reported to have anti-allergic activity. Compounds 1 (X═H,Y═H), 2 (X═H, Y═H), 3 (X═OH, Y═H), 4 (X═OMe, Y═H), 5 (X═OMe, Y═OMe), 6(X═NO₂, Y═H), 7 (X═NH₂, Y═H), (see Table 1, therein) are shown below.

[0010] Berryman et al., 1995, 1997, describe a number of substitutedchalcones which are intermediates used in the preparation of certainfuranone and thiofuranone compounds reported to have activity asendothelin I antagonists.

[0011] Some of the chalcone intermediates have a 3,4-methylenedioxygroup on the A-ring, as shown in the core structure below. See, e.g., inBerryman et al., 1995, Examples 36, 155, 187, 191, 195, 200, 201, 205,209, 213, 217, 224, 232, 238, 242, 246, 263, 268, 280, 287, 288, 289,298, 326, 345, 352, 353, 354, 355, 357, 366, 367, 368, 369, 370, 371,378, 380, 387, 405, and 406; and additionally, in Berryman et al., 1997,Examples 421, 435, and 446. Various B-ring substituents are illustrated,including: 4-hydroxy; 2-methoxy; 3-methoxy; 4-methoxy;2-allyloxy-4-methoxy; 4-isopropoxy; 2,4-dimethoxy; 3,4-dimethoxy;3,4-methylenedioxy; 3,4-methylenedioxy-5-methoxy; and 3,4-ethylenedioxy.

[0012] Although many of the chalcone intermediates have an A-ringsubstituent which is 4-methoxy, one (Example 1, page 55, in Berryman etal., 1995) has a 4-(C₂₋₆alkoxy) substituent, specifically, a4-isopropoxy substituent, as shown below.

[0013] Ikeda Shunichi et al., 1996, describe several substitutedchalcones reported to be active as antitumour agents. Compounds 1 (X═H)(also referred to herein as DMU-103), 2 (X=Me), and 3 (X=Et) (see Table1, therein) are shown below.

[0014] Ducki et al., 1998, describe several substituted chalcones whichwere screened for cytotoxic activity against the human K562 humanleukemia cell line (which does not express CYP1B1). Compounds 2a-d (X═H)and 5a-d (X═Me) (see Table 3, therein) are shown below. The X=Mecompounds were found to be much more active against K562 cells than theX═H compounds (see Table 3 therein), thus favouring the presence of the“ene”-substituent. Compound 2b is also referred to herein as DMU-135.

[0015] Kharazmi et al., 1999, describe a large number of substitutedchalcones alleged to be suitable for the treatment of, inter alia,inflammatory conditions and neoplasias. See, e.g., Example 1 (pages71-94) therein; the ring numbering scheme, shown below, is illustratedat page 132 therein. None of the compounds have a 4-(C₂₋₆alkoxy)substituent or a 3,4-methylenedioxy substituent (using their numberingscheme).

[0016] Potter et al., 1999, 2001 a, describe several 3,4,5-trimethoxychalcones which were shown to inhibit preferentially the growth of cellsexpressing cytochrome P450 enzyme CYP1B1 as compared to cells which donot. Compounds VI (X═OMe, Y═H, Z═H, cis), VIII (X═OMe, Y═H, Z═H, trans),VIII (X═OH, Y═H, Z=H), IX (X═OMe, Y═OMe, Z=H), XI (X═OMe, Y═H, Z=Me) areshown below. Compound VII was reported to be 200-fold more cytotoxic tothe cell line expressing CYP1B1 than to the parental cell line notexpressing this enzyme.

[0017] Potter et al., 2001 b, describes certain substituted1-(4-methoxyphenyl)-3-(3,5-dimethoxyphenyl)prop-1-en-3one of thefollowing general formula, which have therapeutic application, and whichare potent antiproliferative agents and antiinflammatory agents.

[0018] Cushman et al., 1995, describes various stilbene derviatives,which are reported to possess utility as anticancer agents.

[0019] There is a great need for additional antiproliferative agentswhich offer one or more of the following benefits:

[0020] (a) improved activity.

[0021] (b) improved selectivity (e.g., against tumour cells versusnormal cells).

[0022] (c) low cytotoxicity as a prodrug, but yields an active drug invivo;

[0023] (d) complement the activity of other treatments (e.g.,chemotherapeutic agents);

[0024] (e) reduced intensity of undesired side-effects;

[0025] (f) fewer undesired side-effects;

[0026] (g) simpler methods of administration;

[0027] (h) reduction in required dosage amounts;

[0028] (i) reduction in required frequency of administration;

[0029] (j) increased ease of synthesis, purification, handling, storage,etc.;

[0030] (k) reduced cost of synthesis, purification, handling, storage,etc.

[0031] Thus, one aim of the present invention is the provision ofcompounds which are potent antiproliferative agents, e.g., anti-canceragents, which offer one or more of the above benefits.

[0032] The inventors have discovered that certain sub-classes ofsubstituted chalcones, described herein, offer one or more of the abovebenefits, and additionally are surprisingly and unexpectedly more activethan corresponding known analogues.

SUMMARY OF THE INVENTION

[0033] One aspect of the present invention pertains to use of an activecompound, as described herein, for the manufacture of a medicament foruse in the treatment of a proliferative condition.

[0034] In one preferred embodiment, the proliferative condition ischaracterised by cells which express CYP1B1,

[0035] In one preferred embodiment, the proliferative condition ischaracterised by cells which express CYP1B1, where the correspondingnormal cells do not express CYP1B1.

[0036] In one preferred embodiment, the proliferative condition iscancer.

[0037] In one preferred embodiment, the proliferative condition is asolid tumour.

[0038] In one preferred embodiment, the proliferative condition is asolid tumour, and is a cancer of the lung, colon, breast, ovarian,prostate, liver, pancreas, brain, or skin.

[0039] In one preferred embodiment, the proliferative condition is asolid tumour, and is a cancer of the breast.

[0040] Another aspect of the present invention pertains to methods oftreating a proliferative condition in a subject comprising administeringto said subject a therapeutically-effective amount of an activecompound, as described herein.

[0041] Another aspect of the present invention pertains to a compound asdescribed herein for the manufacture of a medicament for use in theprophylactic treatment of a proliferative condition characterised bycells which express CYP1B1.

[0042] Another aspect of the present invention pertains to a method ofprophylactically treating a proliferative condition characterised bycells which express CYP1B1 in a patient comprising administering to saidpatient a therapeutically-effective amount of a compound as describedherein.

[0043] Another aspect of the present invention pertains to use of anactive compound, as described herein, for the manufacture of amedicament for use in the treatment of an inflammatory condition.

[0044] Another aspect of the present invention pertains to a method oftreating an inflammatory condition in a patient comprising administeringto said patient a therapeutically-effective amount of a compound asdescribed herein.

[0045] In one preferred embodiment, the inflammatory condition isrheumatoid arthritis, rheumatic fever, osteoarthritis, inflammatorybowel disease, psoriasis, or bronchial asthma.

[0046] Another aspect of the present invention pertains to a compound asdescribed herein, wherein R^(A3) is —H, for use in a method of diagnosisof the human or animal body. In one preferred embodiment, the diagnosisis for detecting the presence of tumour cells expressing the CYP1B1enzyme.

[0047] Another aspect of the present invention pertains to the use of acompound as described herein, wherein R^(A3) is —H, for detecting thepresence of cells (e.g., tumour cells) expressing the CYP1B1 enzyme.

[0048] Another aspect of the present invention pertains to a method ofdiagnosis of a subject for the presence of cells (e.g., tumour cells)expressing the CYP1B1 enzyme, comprising:

[0049] (a) administering to the patient a compound as described herein,wherein R^(A3) is —H;

[0050] (b) determining the amount of the corresponding hydroxylatedmetabolite, wherein R^(A3) is —OH, which is subsequently produced; and,

[0051] (c) correlating the amount with the presence or absence of thecells (e.g., tumour cells) in the patient.

[0052] Another aspect of the invention pertains to active compounds, asdescribed herein, which treat a proliferative condition, such as cancer.

[0053] Another aspect of the present invention pertains to a compositioncomprising a compound as described herein and a pharmaceuticallyacceptable carrier.

[0054] Another aspect of the present invention pertains to an activecompound, as described herein, for use in a method of treatment of thehuman or animal body by therapy.

[0055] Another aspect of the present invention pertains to an activecompound, as described herein, for use in a method of treatment of aproliferative condition of the human or animal body by therapy.

[0056] Another aspect of the present invention pertains to an activecompound, as described herein, for use in a method of treatment ofcancer of the human or animal body by therapy.

[0057] Another aspect of the present invention pertains to an activecompound, as described herein, for use in a method of treatment of aninflammatory condition of the human or animal body by therapy.

[0058] Another aspect of the present invention pertains to methods ofregulating (e.g., inhibiting) cell proliferation, comprising contactinga cell with an effective amount of an active compound, as describedherein, whether in vitro or in vivo.

[0059] Another aspect of the present invention pertains to a kitcomprising (a) the active compound, preferably provided as apharmaceutical composition and in a suitable container and/or withsuitable packaging; and (b) instructions for use, for example, writteninstructions on how to administer the active compound.

[0060] Another aspect of the present invention pertains to compoundsobtainable by a method of synthesis as described herein, or a methodcomprising a method of synthesis as described herein.

[0061] Another aspect of the present invention pertains to compoundsobtained by a method of synthesis as described herein, or a methodcomprising a method of synthesis as described herein.

[0062] Another aspect of the present invention pertains to novelintermediates, as described herein, which are suitable for use in themethods of synthesis described herein.

[0063] Another aspect of the present invention pertains to the use ofsuch novel intermediates, as described herein, in the methods ofsynthesis described herein.

[0064] As will be appreciated by one of skill in the art, features andpreferred embodiments of one aspect of the invention will also pertainto other aspects of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0065]FIG. 1 is a graph of cell survivial (%) versus concentration (μM)of compound DMU-135, for (A) the TCDD-induced MCF-7 cell line (▪) and(B) the MCF-7 cell line (▾).

[0066]FIG. 2 is a graph of cell survivial (%) versus concentration (pM)of compound DMU-135, for (A) the normal breast cell line MCF-10A (∘),and (B) the advanced breast cancer cell line MDA468 ().

DETAILED DESCRIPTION OF THE INVENTION

[0067] Compounds

[0068] One aspect of the present invention pertains to compounds of thefollowing formula:

[0069] wherein:

[0070] each of R^(B2), R^(B3), R^(B4), and R^(B5) is independently —H,—OH, or —OMe;

[0071] each of R¹ and R² is independently —H, optionally substitutedC₁₋₄alkyl, or optionally substituted C₅₋₂₀aryl;

[0072] R^(A3) is —H, —OH, —OC(═O)R^(E), —OS(═O)₂OH, or —OP(═O)(OH)₂;

[0073] R^(E) is —H, optionally substituted C₁₋₆alkyl, optionallysubstituted C₃₋₂₀heterocyclyl, or optionally substituted C₅₋₂₀aryl;

[0074] and pharmaceutically acceptable salts, solvates, amides, esters,ethers, chemically protected forms, and prodrugs thereof.

[0075] In one embodiment, R^(A3), R^(B2), R^(B3), R^(B4), and R^(B5) areas defined herein, but with the proviso that: if: R^(A3) is —H andR^(B2) is —H and R^(B3) is —OMe and R^(B4) is —OMe; then: R^(B5) is not—OMe (that is, the compound is not a“3′-unsubstituted-3,4,5-trimethoxyphenyl” compound).

[0076] In one embodiment, R^(B2), R^(B3), R^(B4), and R^(B5) are asdefined herein, but with the proviso that: if: R^(B2) is —H and R^(B3)is —OMe and R^(B4) is —OMe; then: R^(B5) is not —OMe (that is, thecompound is not a “3,4,5-trimethoxyphenyl” compound).

[0077] Note that the compounds of the present invention are all of the“E” (entgegen) or “trans” form, that is, the (optionally substituted)4-methoxy-phenyl group (styryl phenyl group) and the3,5-dimethoxybenzoyl group (acyl phenyl group) are positioned “trans”with respect to one another on the carbon-carbon double bond of theprop-1-ene backbone.

[0078] Substituents R^(B2) R^(B3), R^(B4), and R^(B5)

[0079] Each of R^(B2), R^(B3), R^(B4), and R^(B5) is independently —H,—OH, or —OMe.

[0080] In one embodiment, one of R^(B2), R^(B3), R^(B4), and R^(B5) is—OH or —OMe, and the others are —H (“monosubstituted”).

[0081] In one embodiment, two of R^(B2), R^(B3), R^(B4), and R^(B5) is—OH or —OMe, and the others are —H (“disubstituted”).

[0082] In one embodiment, three of R^(B2), R^(B3), R^(B4), and R^(B5) is—OH or —OMe, and the other is —H (“trisubstituted”).

[0083] In one embodiment, each of R^(B2), R^(B3), R^(B4), and R^(B5) is—OH or —OMe (“tetrasubstituted”).

[0084] In one embodiment, one of R^(B2), R^(B3), R^(B4), and R^(B5) is—OMe, and the others are independently —H or —OH (“monomethoxy”).

[0085] In one embodiment, two of R^(B2), R^(B3), R^(B4), and R^(B5) is—OMe, and the others are independently —H or —OH (“dimethoxy”).

[0086] In one embodiment, two of R^(B2), R^(B3), R^(B4), and R^(B5) is—OMe, and the others are independently —H or —OH (“dimethoxy”); and thetwo —OMe groups are not adjacent to each other.

[0087] In one embodiment, two of R^(B2), R^(B3), R^(B4), and R^(B5) is—OMe; one of the others is —OH; and the last is —H(“dimethoxy-hydroxy”).

[0088] In one embodiment, two of R^(B2), R^(B3), R^(B4), and R^(B5) is—OMe; one of the others is —OH; and the last is —H(“dimethoxy-hydroxy”); and the two —OMe groups are not adjacent to eachother.

[0089] In one embodiment, three of R^(B2), R^(B3), R^(B4), and R^(B5) is—OMe, and the others are independently —H or —OH (“trimethoxy”).

[0090] In one embodiment, each of R^(B2), R^(B3), R^(B4), and R^(B5) is—OMe (“tetramethoxy”).

[0091] In one embodiment, each of R^(B2), R^(B3), R^(B4), and R^(B5) isindependently —H or —OMe.

[0092] In one embodiment, one of R^(B2), R^(B3), R^(B4), and R^(B5) is—OMe, and the others are —H (“monosubstituted, monomethoxy”).

[0093] In one embodiment, two of R^(B2), R^(B3), R^(B4), and R^(B5) is—OMe, and the others are —H (“disubstituted, dimethoxy”).

[0094] In one embodiment, two of R^(B2), R^(B3), R^(B4), and R^(B5) is—OMe, and the others are —H (“disubstituted, dimethoxy”); and the two—OMe groups are not adjacent to each other.

[0095] In one embodiment, three of R^(B2), R^(B3), R^(B4), and R^(B5) is—OMe, and the other is —H (“trisubstituted, trimethoxy”).

[0096] In one embodiment, the compound has one of the followingformulae:

[0097] Substituents R¹ and R²

[0098] Each of R¹ and R² is independently —H, optionally substitutedC₁₋₄alkyl, or optionally substituted C₅₋₂₀aryl.

[0099] In one embodiment, one of R¹ and R² is —H; and the other is —H,optionally substituted C₁₋₄alkyl, or optionally substituted C₅₋₂₀aryl.

[0100] In one embodiment, R¹ is —H; and R² is —H, optionally substitutedC₁₋₄alkyl, or optionally substituted C₅₋₂₀aryl.

[0101] In one embodiment, R² is —H; and R¹ is —H, optionally substitutedC₁₋₄alkyl, or optionally substituted C₅₋₂₀aryl.

[0102] In one embodiment, each of R¹ and R² is independently —H, —Me, or—Ph.

[0103] In one embodiment, one of R¹ and R² is —H; and the other is —H,—Me, or —Ph.

[0104] In one embodiment, R¹ is —H; and R² is —H, —Me, or —Ph.

[0105] In one embodiment, R² is —H; and R¹ is —H, —Me, or —Ph.

[0106] In one embodiment, each of R¹ and R² is independently —H or —Me.

[0107] In one embodiment, one of R¹ and R² is —H; and the other is —H or—Me.

[0108] In one embodiment, R¹ is —H; and R² is —H or —Me.

[0109] In one embodiment, R² is —H; and R¹ is —H or —Me.

[0110] In one embodiment, R¹ and R² are both —H:

[0111] In one embodiment, R¹ and R² are both —H and the compound has oneof the following formulae:

[0112] Substituent R^(A3)

[0113] R^(A3) is —H, —OH, —OC(═O)R^(E), —OS(═O)₂OH, or —OP(═O)(OH)₂,wherein R^(E) is —H, optionally substituted C₁₋₆alkyl, optionallysubstituted C₃₋₂₀heterocyclyl, or optionally substituted C₅₋₂₀aryl.

[0114] In one embodiment, R^(E) is selected from: —CH₃ (so that—C(═O)R^(E) is —C(═O)CH₃, acetyl); —CH₂CH₃ (so that —C(═O)RE is—C(═O)CH₂CH₃, propionyl); —C(CH₃)₃ (so that —C(═O)R^(E) is—C(═O)C(CH₃)₃, pivaloyl); and —Ph (so that —C(═O)R^(E) is —C(═O)Ph,benzoyl).

[0115] In one embodiment, R^(A3) is —OC(═O)RE, —OS(═O)₂OH, or—OP(═O)(OH)₂. Such compounds may conveniently be referred to herein as“esterified compounds.”

[0116] In one embodiment, R^(A3) is —H, —OH, or OC(═O)R^(E).

[0117] In one embodiment, R^(A3) is —H or —OH.

[0118] In one embodiment, R^(A3) is —H, as shown below. Such compoundsmay conveniently be referred to herein as “non-hydroxylated compounds.”

[0119] In one embodiment, R^(A3) is —H and the compound has one of thefollowing formulae:

[0120] In one embodiment, R^(A3) is —H; R¹ and R² are both —H; and thecompound has one of the following formulae:

[0121] In one embodiment, R^(A3) is —OH, as shown below. Such compoundsmay conveniently be referred to herein as “hydroxylated compounds.

[0122] In one embodiment, R^(A3) is —OH and the compound has one of thefollowing formulae:

[0123] In one embodiment, R^(A3) is —OH; R¹ and R² are both —H; and thecompound has one of the following formulae:

[0124] Some Specific Embodiments

[0125] Some specific embodiments of the present invention are shownbelow.

[0126] Metabolites

[0127] Another aspect of the present invention pertains to compoundswhich are metabolites of the above-described compounds.

[0128] For example, in one embodiment, the metabolite compounds are onesin which the 4,5-methylenedioxy group has been cleaved and replaced with4,5-dihydroxy groups, as shown below:

[0129] Some specific examples of such metabolites are shown below.

[0130] Chemical Terms

[0131] The term “carbo,” “carbyl,” “hydrocarbon” and “hydrocarbyl,” asused herein, pertain to compounds and/or groups which have only carbonand hydrogen atoms.

[0132] The term “hetero,” as used herein, pertains to compounds and/orgroups which have at least one heteroatom, for example, multivalentheteroatoms (which are also suitable as ring heteroatoms) such as boron,silicon, nitrogen, phosphorus, oxygen, and sulfur, and monovalentheteroatoms, such as fluorine, chlorine, bromine, and iodine.

[0133] The term “saturated,” as used herein, pertains to compoundsand/or groups which do not have any carbon-carbon double bonds orcarbon-carbon triple bonds.

[0134] The term “unsaturated,” as used herein, pertains to compoundsand/or groups which have at least one carbon-carbon double bond orcarbon-carbon triple bond.

[0135] The term “aliphatic,” as used herein, pertains to compoundsand/or groups which are linear or branched, but not cyclic (also knownas “acyclic” or “open-chain” groups).

[0136] The term “cyclic,” as used herein, pertains to compounds and/orgroups which have one ring, or two or more rings (e.g., spiro, fused,bridged).

[0137] The term “ring,” as used herein, pertains to a closed ring offrom 3 to 10 covalently linked atoms, more preferably 3 to 8 covalentlylinked atoms.

[0138] The term “aromatic ring,” as used herein, pertains to a closedring of from 3 to 10 covalently linked atoms, more preferably 5 to 8covalently linked atoms, which ring is aromatic.

[0139] The term “heterocyclic ring,” as used herein, pertains to aclosed ring of from 3 to 10 covalently linked atoms, more preferably 3to 8 covalently linked atoms, wherein at least one of the ring atoms isa multivalent ring heteroatom, for example, nitrogen, phosphorus,silicon, oxygen, and sulfur, though more commonly nitrogen, oxygen, andsulfur.

[0140] The term “alicyclic,” as used herein, pertains to compoundsand/or groups which have one ring, or two or more rings (e.g., spiro,fused, bridged), wherein said ring(s) are not aromatic.

[0141] The term “aromatic,” as used herein, pertains to compounds and/orgroups which have one ring, or two or more rings (e.g., fused), whereinat least one of said ring(s) is aromatic.

[0142] The term “heterocyclic,” as used herein, pertains to cycliccompounds and/or groups which have one heterocyclic ring, or two or moreheterocyclic rings (e.g., spiro, fused, bridged), wherein said ring(s)may be alicyclic or aromatic.

[0143] The term “heteroaromatic,” as used herein, pertains to cycliccompounds and/or groups which have one heterocyclic ring, or two or moreheterocyclic rings (e.g., fused), wherein said ring(s) is aromatic.

[0144] Substituents

[0145] The phrase “optionally substituted,” as used herein, pertains toa parent group which may be unsubstituted or which may be substituted.

[0146] Unless otherwise specified, the term “substituted,” as usedherein, pertains to a parent group which bears one or more substituents.The term “substituent” is used herein in the conventional sense andrefers to a chemical moiety which is covalently attached to, appendedto, or if appropriate, fused to, a parent group. A wide variety ofsubstituents are well known, and methods for their formation andintroduction into a variety of parent groups are also well known.

[0147] In one preferred embodiment, the substituent(s) are independentlyselected from: halo; hydroxy; ether (e.g., C₁₋₇alkoxy); formyl; acyl(e.g., C₁₋₇alkylacyl, C₅₋₂₀arylacyl); acylhalide; carboxy; ester;acyloxy; amido; acylamido; thioamido; tetrazolyl; amino; nitro; nitroso;azido; cyano; isocyano; cyanato; isocyanato; thiocyano; isothiocyano;sulfhydryl; thioether (e.g., C₁₋₇alkylthio); sulfonic acid; sulfonate;sulfone; sulfonyloxy; sulfinyloxy; sulfamino; sulfonamino; sulfinamino;sulfamyl; sulfonamido; C₁₋₇alkyl (including, e.g., C¹⁻⁷haloalkyl,C₁₋₇hydroxyalkyl, C₁₋₇carboxyalkyl, C¹⁻⁷aminoalkyl,C₅₋₂₀aryl-C₁₋₇alkyl); C₃₋₂₀heterocyclyl; or C₅₋₂₀aryl (including, e.g.,C₅₋₂₀carboaryl, C₅₋₂₀heteroaryl, C¹⁻⁷alkyl-C₅₋₂₀aryl andC₅₋₂₀haloaryl)).

[0148] In one preferred embodiment, the substituent(s) are independentlyselected from:

[0149] —F, —Cl, —Br, and —I;

[0150] —OH;

[0151] —OMe, —OEt, —O(tBu), and —OCH₂Ph;

[0152] —SH;

[0153] —SMe, —SEt, —S(tBu), and —SCH₂Ph;

[0154] —C(═O)H;

[0155] —C(—O)Me, —C(═O)Et, —C(═O)(tBu), and —C(═O)Ph;

[0156] —C(═O)OH;

[0157] —C(—O)OMe, —C(═O)OEt, and —C(═O)O(tBu);

[0158] —C(═O)NH₂, —C(═O)NHMe, —C(═O)NMe₂, and —C(═O)NHEt;

[0159] —NHC(═O)Me, —NHC(═O)Et, —NHC(═O)Ph, succinimidyl, and maleimidyl;

[0160] —NH₂, —NHMe, —NHEt, —NH(iPr), —NH(nPr), —NMe₂, —NEt₂, —N(iPr)₂,—N(nPr)₂,

[0161] —N(nBu)₂, and —N(tBu)₂;

[0162] —CN;

[0163] —NO₂;

[0164] -Me, -Et, -nPr, -iPr, -nBu, -tBu;

[0165] —CF₃, —CHF₂, —CH₂F, —CCl₃, —CBr₃, —CH₂CH₂F, —CH₂CHF₂, and—CH₂CF₃;

[0166] —OCF₃, —OCHF₂, —OCH₂F, —OCCl₃, —OCBr₃, —OCH₂CH₂F, —OCH₂CHF₂, and

[0167] —OCH₂CF₃;

[0168] —CH₂OH, —CH₂CH₂OH, and —CH(OH)CH₂OH;

[0169] —CH₂NH₂, —CH₂CH₂NH₂, and —CH₂CH₂NMe₂; and, optionally substitutedphenyl.

[0170] The substituents are described in more detail below.

[0171] C₁₋₇alkyl: The term “C₁₋₇alkyl,” as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from aC₁₋₇hydrocarbon compound having from 1 to 7 carbon atoms, which may bealiphatic or alicyclic, or a combination thereof, and which may besaturated, partially unsaturated, or fully unsaturated.

[0172] Examples of (unsubstituted) saturated linear C₁₋₇alkyl groupsinclude, but are not limited to, methyl, ethyl, n-propyl, n-butyl, andn-pentyl (amyl).

[0173] Examples of (unsubstituted) saturated branched C₁₋₇alkyl groupsinclude, but are not limited to, iso-propyl, iso-butyl, sec-butyl,tert-butyl, and neo-pentyl.

[0174] Examples of saturated alicyclic (also carbocyclic) C¹⁻⁷alkylgroups (also referred to as “C₃₋₇cycloalkyl” groups) include, but arenot limited to, unsubstituted groups such as cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, and norbornane, as well as substituted groups(e.g., groups which comprise such groups), such as methylcyclopropyl,dimethylcyclopropyl, methylcyclobutyl, dimethylcyclobutyl,methylcyclopentyl, dimethylcyclopentyl, methylcyclohexyl,dimethylcyclohexyl, cyclopropylmethyl and cyclohexylmethyl.

[0175] Examples of (unsubstituted) unsaturated C₁₋₇alkyl groups whichhave one or more carbon-carbon double bonds (also referred to as“C₂₋₇alkenyl” groups) include, but are not limited to, ethenyl (vinyl,—CH═CH₂), 2-propenyl (allyl, —CH—CH═CH₂), isopropenyl (—C(CH₃)═CH₂),butenyl, pentenyl, and hexenyl.

[0176] Examples of (unsubstituted) unsaturated C₁₋₇alkyl groups whichhave one or more carbon-carbon triple bonds (also referred to as“C₂₋₇alkynyl” groups) include, but are not limited to, ethynyl (ethinyl)and 2-propynyl (propargyl).

[0177] Examples of unsaturated alicyclic (also carbocyclic) C¹⁻⁷alkylgroups which have one or more carbon-carbon double bonds (also referredto as “C₃₋₇cycloalkenyl” groups) include, but are not limited to,unsubstituted groups such as cyclopropenyl, cyclobutenyl, cyclopentenyl,and cyclohexenyl, as well as substituted groups (e.g., groups whichcomprise such groups) such as cyclopropenylmethyl andcyclohexenylmethyl.

[0178] Additional examples of substituted C₃₋₇cycloalkyl groups include,but are not limited to, those with one or more other rings fusedthereto, for example, those derived from: indene (C₉), indan(2,3-dihydro-1H-indene) (C₉), tetraline (1,2,3,4-tetrahydronaphthalene(C₁₀), adamantane (C₁₀), decalin (decahydronaphthalene) (C₁₂), fluorene(C₁₃), phenalene (C₁₃). For example, 2H-inden-2-yl is a C₅cycloalkylgroup with a substituent (phenyl) fused thereto.

[0179] C₃₋₂₀heterocyclyl: The term “C₃₋₂₀heterocyclyl,” as used herein,pertains to a monovalent moiety obtained by removing a hydrogen atomfrom a ring atom of a C₃₋₂₀heterocyclic compound, said compound havingone ring, or two or more rings (e.g., spiro, fused, bridged), and havingfrom 3 to 20 ring atoms, of which from 1 to 10 are ring heteroatoms, andwherein at least one of said ring(s) is a heterocyclic ring. Preferably,each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ringheteroatoms.

[0180] In this context, the prefixes (e.g., C₃₋₂₀, C₃₋₇, C₅₋₆, etc.)denote the number of ring atoms, or range of number of ring atoms,whether carbon atoms or heteroatoms. For example, the term“C₅₋₆heterocyclyl,” as used herein, pertains to a heterocyclyl grouphaving 5 or 6 ring atoms. Examples of groups of heterocyclyl groupsinclude C₃₋₂₀heterocyclyl, C₃₋₇heterocyclyl, C₅₋₇heterocyclyl.

[0181] Examples of (non-aromatic) monocyclic heterocyclyl groupsinclude, but are not limited to, those derived from:

[0182] N₁: aziridine (C₃), azetidine (C₄), pyrrolidine(tetrahydropyrrole) (C₅), pyrroline (e.g., 3-pyrroline,2,5-dihydropyrrole) (C₅), 2H-pyrrole or 3H-pyrrole (isopyrrole,isoazole) (C₅), piperidine (C₆), dihydropyridine (C₆),tetrahydropyridine (C₆), azepine (C₇);

[0183] O₁: oxirane (C₃), oxetane (C₄), oxolane (tetrahydrofuran) (C₅),oxole (dihydrofuran) (C₅), oxane (tetrahydropyran) (C₆), dihydropyran(C₆), pyran (C₆), oxepin (C₇);

[0184] S₁: thiirane (C₃), thietane (C₄), thiolane (tetrahydrothiophene)(C₅), thiane (tetrahydrothiopyran) (C₆), thiepane (C₇);

[0185] O₂: dioxolane (C₅), dioxane (C₆), and dioxepane (C₇);

[0186] O₃: trioxane (C₆);

[0187] N₂: imidazolidine (C₅), pyrazolidine (diazolidine) (C₅),imidazoline (C₅), pyrazoline (dihydropyrazole) (C5), piperazine (C₆);

[0188] N₁O₁: tetrahydrooxazole (C₅), dihydrooxazole (C₅),tetrahydroisoxazole (C₅), dihydroisoxazole (C₅), morpholine (C₆),tetrahydrooxazine (C₆), dihydrooxazine (C₆), oxazine (C₆);

[0189] N₁S₁: thiazoline (C₅), thiazolidine (C₅), thiomorpholine (C₆);N₂O₁: oxadiazine (C₆);

[0190] O₁S₁: oxathiole (C₅) and oxathiane (thioxane) (C₆); and,

[0191] N₁O₁S₁: oxathiazine (C₆).

[0192] Examples of substituted (non-aromatic) monocyclic heterocyclylgroups include saccharides, in cyclic form, for example, furanoses (C₅),such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse,and pyranoses (C₆), such as allopyranose, altropyranose, glucopyranose,mannopyranose, gulopyranose, idopyranose, galactopyranose, andtalopyranose.

[0193] Examples of heterocyclyl groups which are also heteroaryl groupsare described below with aryl groups.

[0194] C₅₋₂₀aryl: The term “C₅₋₂₀aryl,” as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from an aromaticring atom of a C₅₋₂₀aromatic compound, said compound having one ring, ortwo or more rings (e.g., fused), and having from 5 to 20 ring atoms, andwherein at least one of said ring(s) is an aromatic ring. Preferably,each ring has from 5 to 7 ring atoms. In this context, the prefixes(e.g., C₃₋₂₀, C₅₋₇, C₅₋₆, etc.) denote the number of ring atoms, orrange of number of ring atoms, whether carbon atoms or heteroatoms. Forexample, the term “C₅₋₆aryl,” as used herein, pertains to an aryl grouphaving 5 or 6 ring atoms. Examples of groups of aryl groups includeC₃₋₂₀aryl, C₅₋₇aryl, C₅ aryl.

[0195] The ring atoms may be all carbon atoms, as in “carboaryl groups”(e.g., C₅₋₂₀carboaryl).

[0196] Examples of carboaryl groups include, but are not limited to,those derived from benzene (i.e., phenyl) (C₆), naphthalene (C₁₀),azulene (C₁₀), anthracene (C₁₄), phenanthrene (C₁₄), naphthacene (C₁₈),and pyrene (C₁₆).

[0197] Examples of aryl groups which comprise fused rings, at least oneof which is an aromatic ring, include, but are not limited to, groupsderived from indene (C₉), isoindene (C₉), and fluorene (C₁₃).

[0198] Alternatively, the ring atoms may include one or moreheteroatoms, including but not limited to oxygen, nitrogen, and sulfur,as in “heteroaryl groups.” In this case, the group may conveniently bereferred to as a “C₅₋₂₀heteroaryl” group, wherein “C₅₋₂₀” denotes ringatoms, whether carbon atoms or heteroatoms. Preferably, each ring hasfrom 5 to 7 ring atoms, of which from 0 to 4 are ring heteroatoms.

[0199] Examples of monocyclic heteroaryl groups include, but are notlimited to, those derived from:

[0200] N₁: pyrrole (azole) (C₅), pyridine (azine) (C₆);

[0201] O₁: furan (oxole) (C₅);

[0202] S₁: thiophene (thiole) (C₅);

[0203] N₁O₁: oxazole (C₅), isoxazole (C₅), isoxazine (C₆);

[0204] N₂O₁: oxadiazole (furazan) (C₅);

[0205] N₃O₁: oxatriazole (C₅);

[0206] N₁S₁: thiazole (C₅), isothiazole (C₅);

[0207] N₂: imidazole (1,3-diazole) (C₅), pyrazole (1,2-diazole) (C₅),pyridazine (1,2-diazine) (C₆), pyrimidine (1,3-diazine) (C₆) (e.g.,cytosine, thymine, uracil), pyrazine (1,4-diazine) (C₆);

[0208] N₃: triazole (C₅), triazine (C₆); and,

[0209] N₄: tetrazole (C₅).

[0210] Examples of heterocyclic groups (some of which are alsoheteroaryl groups) which comprise fused rings, include, but are notlimited to:

[0211] C₉heterocyclic groups (with 2 fused rings) derived frombenzofuran (O₁), isobenzofuran (O₁), indole (N₁), isoindole (N₁), purine(N₄) (e.g., adenine, guanine), benzimidazole (N₂), benzoxazole (N₁O¹),benzisoxazole (N₁O₁), benzodioxole (O₂), benzofurazan (N₂O₁),benzotriazole (N₃), benzothiofuran (S₁), benzothiazole (N₁S₁),benzothiadiazole (N₂S);

[0212] C₁₀heterocyclic groups (with 2 fused rings) derived frombenzodioxan (O₂), quinoline (N₁), isoquinoline (N₁), benzoxazine (N₁O₁),benzodiazine (N₂), pyridopyridine (N₂), quinoxaline (N₂), quinazoline(N₂);

[0213] C₁₃heterocyclic groups (with 3 fused rings) derived fromcarbazole (N₁), dibenzofuran (O₁), dibenzothiophene (S₁); and,

[0214] C₁₄heterocyclic groups (with 3 fused rings) derived from acridine(N₁), xanthene (O₁), phenoxathiin (O₁S₁), phenazine (N₂), phenoxazine(N₁O₁), phenothiazine (N₁S₁), thianthrene (S₂), phenanthridine (N₁),phenanthroline (N₂), phenazine (N₂).

[0215] Heterocyclic groups (including heteroaryl groups) which have anitrogen ring atom in the form of an —NH— group may be N-substituted,that is, as —NR—. For example, pyrrole may be N-methyl substituted, togive N-methypyrrole. Examples of N-substitutents include, but are notlimited to C₁₋₇alkyl, C₃₋₂₀heterocyclyl, C₅₋₂₀aryl, and acyl groups.

[0216] Heterocyclic groups (including heteroaryl groups) which have anitrogen ring atom in the form of an —N=group may be substituted in theform of an N-oxide, that is, as —N(→O)=(also denoted —N⁺(→O⁻)=). Forexample, quinoline may be substituted to give quinoline N-oxide;pyridine to give pyridine N-oxide; benzofurazan to give benzofurazanN-oxide (also known as benzofuroxan).

[0217] Cyclic groups may additionally bear one or more oxo (═O) groupson ring carbon atoms. Monocyclic examples of such groups include, butare not limited to, those derived from:

[0218] C₅: cyclopentanone, cyclopentenone, cyclopentadienone;

[0219] C₆: cyclohexanone, cyclohexenone, cyclohexadienone;

[0220] O₁: furanone (C₅), pyrone (C₆);

[0221] N₁: pyrrolidone (pyrrolidinone) (C₅), piperidinone (piperidone)(C₆), piperidinedione (C₆);

[0222] N₂: imidazolidone (imidazolidinone) (C₅), pyrazolone(pyrazolinone) (C₅), piperazinone (C₆), piperazinedione (C₆),pyridazinone (C₆), pyrimidinone (C₆) (e.g., cytosine), pyrimidinedione(C₆) (e.g., thymine, uracil), barbituric acid (C₆);

[0223] N₁S₁: thiazolone (C₅), isothiazolone (C₅);

[0224] N₁O₁: oxazolinone (C₅).

[0225] Polycyclic examples of such groups include, but are not limitedto, those derived from:

[0226] C₉: indenedione;

[0227] N₁: oxindole (C₉);

[0228] O¹.: benzopyrone (e.g., coumarin, isocoumarin, chromone) (C₁₀);

[0229] N₁O₁: benzoxazolinone (C₉), benzoxazolinone (C₁₀);

[0230] N₂: quinazolinedione (C₁₀);

[0231] N₄: purinone (C₉) (e.g., guanine).

[0232] Still more examples of cyclic groups which bear one or more oxo(═O) groups on ring carbon atoms include, but are not limited to, thosederived from:

[0233] cyclic anhydrides (—C(═O)—O—C(═O)— in a ring), including but notlimited to maleic anhydride (C₅), succinic anhydride (C₅), and glutaricanhydride (C₆);

[0234] cyclic carbonates (—O—C(═O)—O— in a ring), such as ethylenecarbonate (C5) and 1,2-propylene carbonate (C₅);

[0235] imides (—C(═O)—NR—C(═O)— in a ring), including but not limitedto, succinimide (C₅), maleimide (C₅), phthalimide, and glutarimide (C6);

[0236] lactones (cyclic esters, —O—C(═O)— in a ring), including, but notlimited to, β-propiolactone, γ-butyrolactone, δ-valerolactone(2-piperidone), and ε-caprolactone;

[0237] lactams (cyclic amides, —NR—C(═O)— in a ring), including, but notlimited to, β-propiolactam (C₄), γ-butyrolactam (2-pyrrolidone) (C₅),δ-valerolactam (C₆), and ε-caprolactam (C₇);

[0238] cyclic carbamates (—O—C(═O)—NR— in a ring), such as 2-oxazolidone(C₅);

[0239] cyclic ureas (—NR—C(═O)—NR— in a ring), such as 2-imidazolidone(C₅) and pyrimidine-2,4-dione (e.g., thymine, uracil) (C₆).

[0240] The above C₁₋₇alkyl, C₃₋₂₀heterocyclyl, and C₅₋₂₀aryl groups,whether alone or part of another substituent, may themselves optionallybe substituted with one or more groups selected from themselves and theadditional substituents listed below.

[0241] Hydrogen: —H. Note that if the substituent at a particularposition is hydrogen, it may be convenient to refer to the compound asbeing “unsubstituted” at that position.

[0242] Halo: —F, —Cl, —Br, and —I.

[0243] Hydroxy: —OH.

[0244] Ether: —OR, wherein R is an ether substituent, for example, aC₁₋₇alkyl group (also referred to as a C₁₋₇alkoxy group, discussedbelow), a C₃₋₂₀heterocyclyl group (also referred to as aC₃₋₂₀hetercyclyloxy group), or a C₅₋₂₀aryl group (also referred to as aC₅₋₂₀aryloxy group), preferably a C₁₋₇alkyl group.

[0245] C₁₋₇alkoxy: —OR, wherein R is a C₁₋₇alkyl group. Examples ofC₁₋₇alkoxy groups include, but are not limited to, —OCH₃ (methoxy),—OCH₂CH₃ (ethoxy) and —OC(CH₃)₃ (tert-butoxy).

[0246] Oxo (keto, -one): ═O. Examples of cyclic compounds and/or groupshaving, as a substituent, an oxo group (═O) include, but are not limitedto, carbocyclics such as cyclopentanone and cyclohexanone;heterocyclics, such as pyrone, pyrrolidone, pyrazolone, pyrazolinone,piperidone, piperidinedione, piperazinedione, and imidazolidone; cyclicanhydrides, including but not limited to maleic anhydride and succinicanhydride; cyclic carbonates, such as propylene carbonate; imides,including but not limited to, succinimide and maleimide; lactones(cyclic esters, —O—C(═O)— in a ring), including, but not limited to,β-propiolactone, γ-butyrolactone, δ-valerolactone, and ε-caprolactone;and lactams (cyclic amides, —NH—C(═O)— in a ring), including, but notlimited to, β-propiolactam, γ-butyrolactam, δ-valerolactam, andε-caprolactam.

[0247] Imino (imine): ═NR, wherein R is an imino substituent, forexample, hydrogen, C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl group, or aC₅₋₂₀aryl group, preferably hydrogen or a C₁₋₇alkyl group. Examples ofimino groups include, but are not limited to, ═NH, ═NMe, ═NEt, and ═NPh.

[0248] Formyl (carbaldehyde, carboxaldehyde): —C(═O)H.

[0249] Acyl (keto): —C(═O)R, wherein R is an acyl substituent, forexample, a C₁₋₇alkyl group (also referred to as C₁₋₇alkylacyl orC₁₋₇alkanoyl), a C₃₋₂₀heterocyclyl group (also referred to asC₃₋₂₀heterocyclylacyl), or a C₅₋₂₀oaryl group (also referred to asC₅₋₂₀arylacyl), preferably a C¹⁻⁷alkyl group. Examples of acyl groupsinclude, but are not limited to, —C(═O)CH₃ (acetyl), —C(═O)CH₂CH₃(propionyl), —C(═O)C(CH₃)₃ (butyryl), and —C(═O)Ph (benzoyl, phenone).

[0250] Acylhalide (haloformyl, halocarbonyl): —C(═O)X, wherein X is —F,—Cl, —Br, or —I, preferably —Cl, —Br, or −I.

[0251] Carboxy (carboxylic acid): —COOH.

[0252] Ester (carboxylate, carboxylic acid ester, oxycarbonyl):—C(═O)OR, wherein R is an ester substituent, for example, a C₁₋₇alkylgroup, a C₃₋₂₀heterocyclyl group, or a C₅₋₂₀aryl group, preferably aC₁₋₇alkyl group. Examples of ester groups include, but are not limitedto, —C(═O)OCH₃, —C(═O)OCH₂CH₃, —C(═O)OC(CH₃)₃, and —C(═O)OPh.

[0253] Acyloxy (reverse ester): —OC(═O)R, wherein R is an acyloxysubstituent, for example, a C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl group,or a C₅₋₂₀aryl group, preferably a C₁₋₇alkyl group. Examples of acyloxygroups include, but are not limited to, —OC(═O)CH₃ (acetoxy),—OC(═O)CH₂CH₃, —OC(═O)C(CH₃)₃, —OC(═O)Ph, and —OC(═O)CH₂Ph.

[0254] Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide):—C(═O)NR¹R², wherein R¹ and R² are independently amino substituents, asdefined for amino groups. Examples of amido groups include, but are notlimited to, —C(═O)NH₂, —C(═O)NHCH₃, —C(═O)NH(CH₃)₂, —C(═O)NHCH₂CH₃, and—C(═O)N(CH₂CH₃)₂, as well as amido groups in which R¹ and R², togetherwith the nitrogen atom to which they are attached, form a heterocyclicstructure as in, for example, piperidinocarbonyl, morpholinocarbonyl,thiomorpholinocarbonyl, and piperazinocarbonyl.

[0255] Acylamido (acylamino): —NR¹C(═O)R², wherein R¹ is an amidesubstituent, for example, a C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl group,or a C₅₋₂₀aryl group, preferably a C₁₋₇alkyl group, and R² is an acylsubstituent, for example, a C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl group,or a C₅₋₂₀aryl group, preferably a C₁₋₇alkyl group. Examples ofacylamido groups include, but are not limited to, —NHC(═O)CH₃,—NHC(═O)CH₂CH₃, and —NHC(═O)Ph. R¹ and R² may together form a cyclicstructure, as in, for example, for example, succinimidyl, maleimidyl,and phthalimidyl:

[0256] Thioamido (thiocarbamyl): —C(═S)NR¹R², wherein R¹ and R² areindependently amino substituents, as defined for amino groups. Examplesof amido groups include, but are not limited to, —C(═S)NH₂, —C(═S)NHCH₃,—C(═S)NH(CH₃)₂, and —C(═S)NHCH₂CH₃.

[0257] Tetrazolyl: a five membered aromatic ring having four nitrogenatoms and one carbon atom,

[0258] Amino: —NR¹R², wherein R¹ and R² are independently aminosubstituents, for example, hydrogen, a C₁₋₇alkyl group (also referred toas C₁₋₇alkylamino or di-C₁₋₇alkylamino), a C₃₋₂₀heterocyclyl group, or aC₅₋₂₀aryl group, preferably H or a C₁₋₇alkyl group, or, in the case of a“cyclic” amino group, R¹ and R², taken together with the nitrogen atomto which they are attached, form a heterocyclic ring having from 4 to 8ring atoms. Examples of amino groups include, but are not limited to,—NH₂, —NHCH₃, —NHCH(CH₃)₂, —N(CH₃)₂, —N(CH₂CH₃)₂, and —NHPh.

[0259] Examples of cyclic amino groups include, but are not limited to,aziridino, azetidino, piperidino, piperazino, morpholino, andthiomorpholino.

[0260] Nitro: —NO₂.

[0261] Nitroso: —NO.

[0262] Azido: —N₃.

[0263] Cyano (nitrile, carbonitrile): —CN.

[0264] Isocyano: —NC.

[0265] Cyanato: —OCN.

[0266] Isocyanato: —NCO.

[0267] Thiocyano (thiocyanato): —SCN.

[0268] Isothiocyano (isothiocyanato): —NCS.

[0269] Sulfhydryl (thiol, mercapto): —SH.

[0270] Thioether (sulfide): —SR, wherein R is a thioether substituent,for example, a C₁₋₇alkyl group (also referred to as a C₁₋₇alkylthiogroup), a C₃₋₂₀heterocyclyl group, or a C₅₋₂₀aryl group, preferably aC₁₋₇alkyl group. Examples of C₁₋₇alkylthio groups include, but are notlimited to, —SCH₃ and —SCH₂CH₃.

[0271] Sulfonic acid (sulfo): —S(═O)₂OH.

[0272] Sulfonate (sulfonic acid ester): —S(═O)₂OR, wherein R is asulfonate substituent, for example, a C₁₋₇alkyl group, aC₃₋₂₀heterocyclyl group, or a C₅₋₂₀aryl group, preferably a C₁₋₇alkylgroup. Examples of sulfonate groups include, but are not limited to,—S(═O)₂OCH₃ and —S(═O)₂OCH₂CH₃.

[0273] Sulfone (sulfonyl): —S(═O)₂R, wherein R is a sulfone substituent,for example, a C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl group, or aC₅₋₂₀aryl group, preferably a C₁₋₇alkyl group. Examples of sulfonegroups include, but are not limited to, —S(═O)₂CH₃ (methanesulfonyl,mesyl), —S(═O)₂CF₃, —S(═O)₂CH₂CH₃, and 4-methylphenylsulfonyl (tosyl).

[0274] Sulfonyloxy: —OS(═O)₂R, wherein R is a sulfonyloxy substituent,for example, a C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl group, or aC₅₋₂₀aryl group, preferably a C₁₋₇alkyl group. Examples of sulfonyloxygroups include, but are not limited to, —OS(═O)₂CH₃ and —OS(═O)₂CH₂CH₃.

[0275] Sulfinyloxy: —OS(═O)R, wherein R is a sulfinyloxy substituent,for example, a C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl group, or aC₅₋₂₀aryl group, preferably a C₁₋₇alkyl group. Examples of sulfinyloxygroups include, but are not limited to, —OS(═O)CH₃ and —OS(═O)CH₂CH₃.

[0276] Sulfamino: —NR¹S(═O)₂OH, wherein R¹ is an amino substituent, asdefined for amino groups. Examples of sulfamino groups include, but arenot limited to, —NHS(═O)₂OH and —N(CH₃)S(═O)₂OH.

[0277] Sulfonamino: —NR¹S(═O)₂R, wherein R¹ is an amino substituent, asdefined for amino groups, and R is a sulfonamino substituent, forexample, a C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl group, or a C₅₋₂₀arylgroup, preferably a C₁₋₇alkyl group. Examples of sulfonamino groupsinclude, but are not limited to, —NHS(═O)₂CH₃ and —N(CH₃)S(═O)₂C₆H₅.

[0278] Sulfinamino: —NR¹S(═O)R, wherein R¹ is an amino substituent, asdefined for amino groups, and R is a sulfinamino substituent, forexample, a C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl group, or a C₅₋₂₀arylgroup, preferably a C₁₋₇alkyl group.

[0279] Examples of sulfinamino groups include, but are not limited to,—NHS(═O)CH₃ and —N(CH₃)S(═O)C₆H₅.

[0280] Sulfamyl: —S(═O)NR¹R² ₁ wherein R¹ and R² are independently aminosubstituents, as defined for amino groups. Examples of sulfamyl groupsinclude, but are not limited to, —S(═O)NH₂, —S(═O)NH(CH₃),—S(═O)N(CH₃)₂, —S(═O)NH(CH₂CH₃), —S(═O)N(CH₂CH₃)₂, and —S(═O)NHPh.

[0281] Sulfonamido: —S(═O)₂NR¹R², wherein R¹ and R² are independentlyamino substituents, as defined for amino groups. Examples of sulfonamidogroups include, but are not limited to, —S(═O)₂NH₂, —S(═O)₂NH(CH₃),—S(═O)₂N(CH₃)₂, —S(═O)₂NH(CH₂CH₃), —S(═O)₂N(CH₂CH₃)₂, and —S(═O)₂NHPh.

[0282] As mentioned above, a C₁₋₇alkyl group may be substituted with,for example, hydroxy (also referred to as a C₁₋₇hydroxyalkyl group),C₁₋₇alkoxy (also referred to as a C₁₋₇alkoxyalkyl group), amino (alsoreferred to as a C₁₋₇aminoalkyl group), halo (also referred to as aC₁₋₇haloalkyl group), carboxy (also referred to as a C₁₋₇carboxyalkylgroup), and C₅₋₂₀aryl (also referred to as a C₅₋₂₀aryl-C₁₋₇alkyl group).

[0283] Similarly, a C₅₋₂₀aryl group may be substituted with, forexample, hydroxy (also referred to as a C₅₋₂₀hydroxyaryl group), halo(also referred to as a C₅₋₂₀haloaryl group), amino (also referred to asa C₅₋₂₀aminoaryl group, e.g., as in aniline), C₁₋₇alkyl (also referredto as a C₁₋₇alkyl-C₅₋₂₀aryl group, e.g., as in toluene), and C₁₋₇alkoxy(also referred to as a C₁₋₇alkoxy-C₅₋₂₀aryl group, e.g., as in anisole).

[0284] These and other specific examples of such substituted groups arealso discussed below.

[0285] C₁₋₇haloalkyl group: The term “C₁₋₇haloalkyl group,” as usedherein, pertains to a C₁₋₇alkyl group in which at least one hydrogenatom (e.g., 1, 2, 3) has been replaced with a halogen atom (e.g., F, Cl,Br, I). If more than one hydrogen atom has been replaced with a halogenatom, the halogen atoms may independently be the same or different.Every hydrogen atom may be replaced with a halogen atom, in which casethe group may conveniently be referred to as a C₁₋₇perhaloalkyl group.”Examples of C₁₋₇haloalkyl groups include, but are not limited to, —CF₃,—CHF₂, —CH₂F, —CCl₃, —CBr₃, —CH₂CH₂F, —CH₂CHF₂, and —CH₂CF₃.

[0286] C₁₋₇hydroxyalkyl: The term “C₁₋₇hydroxyalkyl group,” as usedherein, pertains to a C₁₋₇alkyl group in which at least one hydrogenatom has been replaced with a hydroxy group. Examples ofC₁₋₇hydroxyalkyl groups include, but are not limited to, —CH₂OH,—CH₂CH₂OH, and —CH(OH)CH₂OH.

[0287] C₁₋₇carboxyalkyl: The term “C₁₋₇carboxyalkyl group,” as usedherein, pertains to a C₁₋₇alkyl group in which at least one hydrogenatom has been replaced with a carboxy group. Examples ofC₁₋₇carboxyalkyl groups include, but are not limited to, —CH₂COOH and—CH₂CH₂COOH.

[0288] C₁₇aminoalkyl: The term “C₁₇aminoalkyl group,” as used herein,pertains to a C₁₋₇alkyl group in which at least one hydrogen atom hasbeen replaced with an amino group. Examples of C₁₋₇aminoalkyl groupsinclude, but are not limited to, —CH₂NH₂, —CH₂CH₂NH₂, and—CH₂CH₂N(CH₃)₂.

[0289] C₁₋₇alkyl-C₅₋₂₀aryl: The term “C₁₋₇alkyl-C₅₋₂₀aryl,” as usedherein, describes certain C₅₋₂₀aryl groups which have been substitutedwith a C₁₋₇alkyl group. Examples of such groups include, but are notlimited to, tolyl (as in toluene), xylyl (as in xylene), mesityl (as inmesitylene), styryl (as in styrene), and cumenyl (as in cumene).

[0290] C₅₋₂₀aryl-C₁₋₇alkyl: The term “C₅₋₂₀aryl-C₁₋₇alkyl,” as usedherein, describers certain C₁₋₇alkyl groups which have been substitutedwith a C₅₋₂₀aryl group. Examples of such groups include, but are notlimited to, benzyl (phenylmethyl), tolylmethyl, phenylethyl, andtriphenylmethyl (trityl).

[0291] C₅₋₂₀haloaryl: The term “C₅₋₂₀haloaryl,” as used herein,describes certain C₅₋₂₀aryl groups which have been substituted with oneor more halo groups. Examples of such groups include, but are notlimited to, halophenyl (e.g., fluorophenyl, chlorophenyl, bromophenyl,or iodophenyl, whether ortho-, meta-, or para-substituted),dihalophenyl, trihalophenyl, tetrahalophenyl, and pentahalophenyl.

[0292] Bidentate Substituents

[0293] Some substituents are bidentate, that is, have two points forcovalent attachment. For example, a bidentate group may be covalentlybound to two different atoms on two different groups, thereby acting asa linker therebetween. Alternatively, a bidentate group may becovalently bound to two different atoms on the same group, therebyforming, together with the two atoms to which it is attached (and anyintervening atoms, if present) a cyclic or ring structure. In this way,the bidentate substituent may give rise to a heterocyclic group/compoundand/or an aromatic group/compound. Typically, the ring has from 3 to 8ring atoms, which ring atoms are carbon or divalent heteroatoms (e.g.,boron, silicon, nitrogen, phosphorus, oxygen, and sulfur, typicallynitrogen, oxygen, and sulfur), and wherein the bonds between said ringatoms are single or double bonds, as permitted by the valencies of thering atoms. Typically, the bidentate group is covalently bound tovicinal atoms, that is, adjacent atoms, in the parent group.

[0294] C₁₋₇alkylene: The term “C₁₋₇alkylene,” as used herein, pertainsto a bidentate moiety obtained by removing two hydrogen atoms, eitherboth from the same carbon atom, or one from each of two different carbonatoms, of a C₁₋₇hydrocarbon compound having from 1 to 7 carbon atoms,which may be aliphatic or alicyclic, or a combination thereof, and whichmay be saturated, partially unsaturated, or fully unsaturated.

[0295] Examples of linear saturated C₁₋₇alkylene groups include, but arenot limited to, —(CH₂)_(n)— where n is an integer from 1 to 7, forexample, —CH₂— (methylene), —CH₂CH₂— (ethylene), —CH₂CH₂CH₂—(propylene), and —CH₂CH₂CH₂CH₂-(butylene).

[0296] Examples of branched saturated C₁₋₇alkylene groups include, butare not limited to, —CH(CH₃)—, —CH(CH₃)CH₂—, —CH(CH₃)CH₂CH₂—,—CH(CH₃)CH₂CH₂CH₂—, —CH₂CH(CH₃)CH₂—, —CH₂CH(CH₃)CH₂CH₂—, —CH(CH₂CH₃)—,—CH(CH₂CH₃)CH₂—, and —CH₂CH(CH₂CH₃)CH₂—.

[0297] Examples of linear partially unsaturated C₁₋₇alkylene groupsinclude, but are not limited to, —CH═CH— (vinylene), —CH═CH—CH₂—,—CH═CH—CH₂—CH₂—, —CH═CH—CH₂—CH₂—CH₂—, —CH═CH—CH═CH—, —CH═CH—CH═CH—CH₂—,—CH═CH—CH═CH—CH₂—CH₂—, —CH═CH—CH₂—CH═CH—, and —CH═CH—CH₂—CH₂—CH═CH—.

[0298] Examples of branched partially unsaturated C₁₋₇alkylene groupsinclude, but are not limited to, —C(CH₃)═CH—, —C(CH₃)═CH—CH₂—, and—CH═CH—CH(CH₃)—.

[0299] Examples of alicyclic saturated C₁₋₇alkylene groups include, butare not limited to, cyclopentylene (e.g., cyclopent-1,3-ylene), andcyclohexylene (e.g., cyclohex-1,4-ylene).

[0300] Examples of alicyclic partially unsaturated C₁₋₇alkylene groupsinclude, but are not limited to, cyclopentenylene (e.g.,4-cyclopenten-1,3-ylene), cyclohexenylene (e.g., 2-cyclohexen-1,4-ylene,3-cyclohexen-1,2-ylene, 2,5-cyclohexadien-1,4-ylene).

[0301] C₅₋₂₀arylene: The term “C₅₋₂₀arylene,” as used herein, pertainsto a bidentate moiety obtained by removing two hydrogen atoms, one fromeach of two different ring atoms of a C₅₋₂₀aromatic compound, saidcompound having one ring, or two or more rings (e.g., fused), and havingfrom 5 to 20 ring atoms, and wherein at least one of said ring(s) is anaromatic ring. Preferably, each ring has from 5 to 7 ring atoms.

[0302] The ring atoms may be all carbon atoms, as in “carboarylenegroups,” in which case the group may conveniently be referred to as a“C₅₋₂₀carboarylene” group.

[0303] Alternatively, the ring atoms may include one or moreheteroatoms, including but not limited to oxygen, nitrogen, and sulfur,as in “heteroarylene groups.” In this case, the group may convenientlybe referred to as a “C₅₋₂₀heteroarylene” group, wherein “C₅₋₂₀” denotesring atoms, whether carbon atoms or heteroatoms. Preferably, each ringhas from 5 to 7 ring atoms, of which from 0 to 4 are ring heteroatoms.

[0304] Examples of C₅₋₂₀arylene groups which do not have ringheteroatoms (i.e., C₅₋₂₀carboarylene groups) include, but are notlimited to, those derived from benzene (i.e., phenyl) (C₆), naphthalene(C₁₀), anthracene (C₁₄), phenanthrene (C₁₄), and pyrene (C₁₆).

[0305] Examples of C₅₋₂₀heteroarylene groups include, but are notlimited to, C₅heteroarylene groups derived from furan (oxole), thiophene(thiole), pyrrole (azole), imidazole (1,3-diazole), pyrazole(1,2-diazole), triazole, oxazole, isoxazole, thiazole, isothiazole,oxadiazole, and oxatriazole; and C₆heteroarylene groups derived fromisoxazine, pyridine (azine), pyridazine (1,2-diazine), pyrimidine(1,3-diazine; e.g., cytosine, thymine, uracil), pyrazine (1,4-diazine),triazine, tetrazole, and oxadiazole (furazan).

[0306] C₅₋₂₀Arylene-C₁₋₇alkylene: The term “C₅₋₂₀arylene-C₁₋₇alkylene,”as used herein, pertains to a bidentate moiety comprising a C₅₋₂₀arylenemoiety, -Arylene-, linked to a C₁₋₇alkylene moiety, -Alkylene-, that is,-Arylene-Alkylene-.

[0307] Examples of C₅₋₂₀arylene-C₁₋₇alkylene groups include, but are notlimited to, phenylene-methylene, phenylene-ethylene,phenylene-propylene, and phenylene-ethenylene (also known asphenylene-vinylene).

[0308] C₅₋₂₀Alkylene-C₁₋₇arylene: The term “C₅₋₂₀alkylene-C₁₋₇arylene,”as used herein, pertains to a bidentate moiety comprising aC₅₋₂₀alkylene moiety, -Alkylene-, linked to a C₁₋₇arylene moiety,-Arylene-, that is, -Alkylene-Arylene-.

[0309] Examples of C₅₋₂₀alkylene-C₁₋₇arylene groups include, but are notlimited to, methylene-phenylene, ethylene-phenylene,propylene-phenylene, and ethenylene-phenylene (also known asvinylene-phenylene).

[0310] Included in the above are the well known ionic, salt, solvate(e.g., hydrate), and protected forms of these substituents. For example,a reference to carboxylic acid (—COOH) also includes carboxylate(—COO⁻). Similarly, a reference to an amino group includes a salt, forexample, a hydrochloride salt, of the amino group. A reference to ahydroxyl group also includes conventional protected forms of a hydroxylgroup. Similarly, a reference to an amino group also includesconventional protected forms of an amino group.

[0311] Acronyms

[0312] For convenience, many chemical moieties are represented hereinusing well known abbreviations, including but not limited to, methyl(Me), ethyl (Et), n-propyl (nPr), iso-propyl (iPr), n-butyl (nBu),tert-butyl (tBu), n-hexyl (nHex), cyclohexyl (cHex), phenyl (Ph),biphenyl (biPh), benzyl (Bn), naphthyl (naph), methoxy (MeO), ethoxy(EtO), benzoyl (Bz), and acetyl (Ac).

[0313] For convenience, many chemical compounds are represented hereinusing well known abbreviations, including but not limited to, methanol(MeOH), ethanol (EtOH), iso-propanol (i-PrOH), methyl ethyl ketone(MEK), acetic acid (AcOH), dichloromethane (methylene chloride, DCM),trifluoroacetic acid (TFA), dimethylformamide (DMF), and tetrahydrofuran(THF).

[0314] Isomers, Salts, Solvates, Protected Forms, and Prodrugs A certaincompound may exist in one or more particular geometric, optical,enantiomeric, diasteriomeric, epimeric, stereoisomeric, tautomeric,conformational, or anomeric forms, including but not limited to, cis-and trans-forms; E- and Z-forms; c-, t-, and r-forms; endo- andexo-forms; R-, S-, and meso-forms; D- and L-forms; (+) and (−) forms;keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- andanticlinal-forms; α- and β-forms; axial and equatorial forms; boat-,chair-, twist-, envelope-, and halfchair-forms; and combinationsthereof, hereinafter collectively referred to as “isomers” (or “isomericforms”).

[0315] Note that, except as discussed below for tautomeric forms,specifically excluded from the term “isomers,” as used herein, arestructural (or constitutional) isomers (i.e., isomers which differ inthe connections between atoms rather than merely by the position ofatoms in space). For example, a reference to a methoxy group, —OCH₃, isnot to be construed as a reference to its structural isomer, ahydroxymethyl group, —CH₂OH. Similarly, a reference toortho-chlorophenyl is not to be construed as a reference to itsstructural isomer, meta-chlorophenyl.

[0316] However, a reference to a class of structures may well includestructurally isomeric forms falling within that class (e.g., C₁₋₇alkylincludes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, andtert-butyl; methoxyphenyl includes ortho-, meta-, andpara-methoxyphenyl).

[0317] The above exclusion does not pertain to tautomeric forms, forexample, keto-, enol-, and enolate-forms, as in, for example, thefollowing tautomeric pairs: keto/enol (illustrated below),imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime,thioketonelenethiol, N-nitroso/hydroxyazo, and nitro/aci-nitro.

[0318] Note that specifically included in the term “isomer” arecompounds with one or more isotopic substitutions. For example, H may bein any isotopic form, including ¹H, ²H (D), and ³H (T); C may be in anyisotopic form, including ¹²C, ¹³C, and ¹⁴C; O may be in any isotopicform, including ¹⁶O and ¹⁸O; and the like.

[0319] Unless otherwise specified, a reference to a particular compoundincludes all such isomeric forms, including racemic and other mixturesthereof. Methods for the preparation (e.g., asymmetric synthesis) andseparation (e.g., fractional crystallisation and chromatographic means)of such isomeric forms are either known in the art or are readilyobtained by adapting the methods taught herein in a known manner.

[0320] Unless otherwise specified, a reference to a particular compoundalso includes ionic, salt, solvate (e.g., hydrate), protected forms, andprodrugs thereof, for example, as discussed below.

[0321] It may be convenient or desirable to prepare, purify, and/orhandle a corresponding salt of the active compound, for example, apharmaceutically-acceptable salt. Examples of pharmaceuticallyacceptable salts are discussed in Berge et al., 1977, “PharmaceuticallyAcceptable Salts,” J. Pharm. Sci., Vol. 66, pp. 1-19.

[0322] For example, if the compound is anionic, or has a functionalgroup which may be anionic (e.g., —COOH may be —COO⁻), then a salt maybe formed with a suitable cation. Examples of suitable inorganic cationsinclude, but are not limited to, alkali metal ions such as Na+ and K+,alkaline earth cations such as Ca²⁺ and Mg²⁺, and other cations such asAl⁺³. Examples of suitable organic cations include, but are not limitedto, ammonium ion (i.e., NH₄ ⁺) and substituted ammonium ions (e.g.,NH₃R⁺, NH₂R₂ ⁺, NHR₃ ⁺, NR₄ ⁺). Examples of some suitable substitutedammonium ions are those derived from: ethylamine, diethylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine. An example ofa common quaternary ammonium ion is N(CH₃)₄ ⁺.

[0323] If the compound is cationic, or has a functional group which maybe cationic (e.g., —NH₂ may be —NH₃ ⁺), then a salt may be formed with asuitable anion. Examples of suitable inorganic anions include, but arenot limited to, those derived from the following inorganic acids:hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric,nitrous, phosphoric, and phosphorous. Examples of suitable organicanions include, but are not limited to, anions from the followingorganic acids: acetic, propionic, succinic, gycolic, stearic, lactic,malic, tartaric, citric, ascorbic, maleic, hydroxymaleic, phenylacetic,glutamic, benzoic, salicylic, sulfanilic, 2-acetyoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethanesulfonic, ethane disulfonic,oxalic, isethionic, and valeric.

[0324] It may be convenient or desirable to prepare, purify, and/orhandle a corresponding solvate of the active compound. The term“solvate” is used herein in the conventional sense to refer to a complexof solute (e.g., active compound, salt of active compound) and solvent.If the solvent is water, the solvate may be conveniently referred to asa hydrate, for example, a mono-hydrate, a di-hydrate, a tri-hydrate,etc.

[0325] It may be convenient or desirable to prepare, purify, and/orhandle the active compound in a chemically protected form. The term“chemically protected form,” as used herein, pertains to a compound inwhich one or more reactive functional groups are protected fromundesirable chemical reactions, that is, are in the form of a protectedor protecting group (also known as a masked or masking group).

[0326] By protecting a reactive functional group, reactions involvingother unprotected reactive functional groups can be performed, withoutaffecting the protected group; the protecting group may be removed,usually in a subsequent step, without substantially affecting theremainder of the molecule. See, for example, Protective Groups inOrganic Synthesis (T. Green and P. Wuts, Wiley, 1991), and ProtectiveGroups in Organic Synthesis (T. Green and P. Wuts; 3rd Edition; JohnWiley and Sons, 1999).

[0327] For example, a hydroxy group may be protected as an ether (—OR)or an ester (—OC(═O)R), for example, as: a t-butyl ether; a benzyl,benzhydryl (diphenylmethyl), or trityl (triphenylmethyl) ether; atrimethylsilyl or t-butyidimethylsilyl ether; or an acetyl ester(—OC(═O)CH₃, —OAc).

[0328] For example, an aldehyde or ketone group may be protected as anacetal or ketal, respectively, in which the carbonyl group (>C═O) isconverted to a diether (>C(OR)₂), by reaction with, for example, aprimary alcohol. The aldehyde or ketone group is readily regenerated byhydrolysis using a large excess of water in the presence of acid.

[0329] For example, an amine group may be protected, for example, as anamide (—NRCO—R) or a urethane (—NRCO—OR), for example, as: a methylamide (—NHCO—CH₃); a benzyloxy amide (—NHCO—OCH₂C₆H₅, —NH-Cbz); as at-butoxy amide (—NHCO—OC(CH₃)₃, —NH-Boc); a 2-biphenyl-2-propoxy amide(—NHCO—OC(CH₃)₂C₆H₄C₆H₅, —NH-Bpoc), as a 9-fluorenylmethoxy amide(—NH-Fmoc), as a 6-nitroveratryloxy amide (—NH-Nvoc), as a2-trimethylsilylethyloxy amide (—NH-Teoc), as a 2,2,2-trichloroethyloxyamide (—NH-Troc), as an allyloxy amide (—NH-Alloc), as a2(-phenylsulfonyl)ethyloxy amide (—NH-Psec); or, in suitable cases(e.g., cyclic amines), as a nitroxide radical (>N—O.).

[0330] For example, a carboxylic acid group may be protected as an esteror an amide, for example, as: a benzyl ester; a t-butyl ester; a methylester; or a methyl amide.

[0331] For example, a thiol group may be protected as a thioether (—SR),for example, as: a benzyl thioether; an acetamidomethyl ether(—S—CH₂NHC(═O)CH₃).

[0332] It may be convenient or desirable to prepare, purify, and/orhandle the active compound in the form of a prodrug. The term “prodrug,”as used herein, pertains to a compound which, when metabolised, yieldsthe desired active compound. Typically, the prodrug is inactive, or lessactive than the active compound, but may provide advantageous handling,administration, or metabolic properties. For example, some prodrugs areesters of the active compound; during metabolysis, the ester group iscleaved to yield the active drug. Also, some prodrugs are activatedenzymatically to yield the active compound, or a compound which, uponfurther chemical reaction, yields the active compound. For example, theprodrug may be a sugar derivative or other glycoside conjugate, or maybe an amino acid ester derivative.

[0333] Synthesis

[0334] Several methods for the chemical synthesis of compounds of thepresent invention are described herein. These methods may be modifiedand/or adapted in known ways in order to facilitate the synthesis ofadditional compounds within the scope of the present invention.

[0335] The compounds of the present invention may be prepared, forexample, by Aldol condensation of the corresponding carbonyl compounds Aand B, as illustrated below in Scheme 2.

[0336] When R¹ is —H, the first compound is a piperonal. When R² is —H,the second compound is an acetophenone.

[0337] Many suitable starting reagents are commercially available (e.g.,from Sigma-Aldrich). Additional reagents may be synthesised using knownmethods, or by modifying known methods in known ways.

[0338] For example, compound DMU-135 may be prepared by stirring amixture of piperonal (A) and 3,4,5-trimethoxyacetophenone (B) in asuitable solvent, e.g., methanol, with added base catalyst, e.g.,aqueous sodium hydroxide for 18 hours at ambient temperature. Thereaction is illustrated below in Scheme 3.

[0339] Compounds for which R^(A3) is —OC(═O)R^(E), —OS(═O)₂OH, or—OP(═O)(OH)₂ may be prepared from their hydroxy analogs (where R^(A3) is—OH) by reaction with an organic acid (i.e., R^(E)COOH) or an inorganicacid (i.e., sulfuric acid, H₂SO₄; phosphoric acid, H₃PO₄).

[0340] The groups —OS(═O)₂OH and —OP(═O)(OH)₂ may be present as such, intheir free acid form, or they may be present as a salt or ester thereof,as discussed above. For example, the group —OS(═O)₂OH may be present as—OS(═O)₂O⁻M⁺, wherein M⁺ is a suitable cation. Similarly, the group—OP(═O)(OH)₂ may be present as —OP(═O)(OH)O M+ or —OP(═O)(O⁻)₂(M⁺)₂,wherein M⁺ is a suitable cation. Examples of suitable cations arediscussed above. In one embodiment, the group —OP(═O)(OH)₂ is present asthe disodium salt, —OP(═O)(O⁻)₂(Na⁺)₂. Other salts and esters aredescribed in Pettit et al, 1995.

[0341] Uses

[0342] The present invention provides active compounds which are capableof regulating (e.g., inhibiting) cell proliferation, as well as methodsof regulating (e.g., inhibiting) cell proliferation, comprisingcontacting a cell with an effective amount of an active compound,whether in vitro or in vivo.

[0343] The term “active,” as used herein, pertains to compounds whichare capable of regulating (e.g., inhibiting) cell proliferation, andspecifically includes both compounds with intrinsic activity (drugs) aswell as prodrugs of such compounds, which prodrugs may themselvesexhibit little or no intrinsic activity.

[0344] One of ordinary skill in the art is readily able to determinewhether or not a candidate compound is active, that is, capable ofregulating (e.g., inhibiting) cell proliferation. For example, assayswhich may conveniently be used to assess the proliferation regulationoffered by a particular compound are described in the examples below.

[0345] For example, a sample of cells (e.g., from a tumour) may be grownin vitro and a candidate compound brought into contact with the cells,and the effect of the compound on those cells observed. As examples of“effect,” the morphological status of the cells may be determined (e.g.,alive or dead). Where the candidate compound is found to exert aninfluence on the cells, this may be used as a prognostic or diagnosticmarker of the efficacy of the compound in methods of treating a patientcarrying cells of the same type (e.g., the tumour or a tumour of thesame cellular type).

[0346] In one aspect, the present invention provides antiproliferativeagents. The term “antiproliferative agent” as used herein, pertains to acompound which treats a proliferative condition (i.e., a compound whichis useful in the treatment of a proliferative condition).

[0347] The terms “cell proliferation,” “proliferative condition,”“proliferative disorder,” and “proliferative disease,” are usedinterchangeably herein and pertain to an unwanted or uncontrolledcellular proliferation of excessive or abnormal cells which isundesired, such as, neoplastic or hyperplastic growth, whether in vitroor in vivo. Examples of proliferative conditions include, but are notlimited to, pre-malignant and malignant cellular proliferation,including but not limited to, malignant neoplasms and tumours, cancers,leukemias, psoriasis, bone diseases, fibroproliferative disorders (e.g.,of connective tissues), and atherosclerosis. Any type of cell may betreated, including but not limited to, lung, colon, breast, ovarian,prostate, liver, pancreas, brain, and skin.

[0348] In another embodiment, the proliferative condition is a solidtumour. In another embodiment, the proliferative condition is a solidtumour, and is a cancer of the lung, colon, breast, ovarian, prostate,liver, pancreas, brain, or skin. In another embodiment, theproliferative condition is a solid tumour, and is a cancer of thebreast.

[0349] As discussed below (see “Prodrugs”), compounds of the presentinvention may act as prodrugs useful as antiproliferative agents withlow intrinsic toxicity, for treatment of proliferative conditions whichare characterised by cells which express the CYP1B1 enzyme.

[0350] Additionally, compounds of the present invention may act asprodrugs useful as selective antiproliferative agents with low intrinsictoxicity, for treatment of proliferative conditions which arecharacterised by cells which express the CYP1B1 enzyme, where thecorresponding normal cells do not express the CYP1B1 enzyme.

[0351] Thus, in one preferred embodiment, the proliferative condition ischaracterised by cells which express CYP1B1. In one preferredembodiment, the proliferative condition is characterised by cells whichexpress CYP1B1, where the corresponding normal cells do not expressCYP1B1. For example, the proliferative condition may be a tumourcharacterised by tumour cells which express CYP1B1, where thecorresponding normal cells do not.

[0352] Antiproliferative compounds of the present invention haveapplication in the treatment of cancer, and so the present inventionfurther provides anticancer agents. The term “anticancer agent” as usedherein, pertains to a compound which treats a cancer (i.e., a compoundwhich is useful in the treatment of a cancer). The anti-cancer effectmay arise through one or more mechanisms, including but not limited to,the regulation of cell proliferation, the inhibition of angiogenesis(the formation of new blood vessels), the inhibition of metastasis (thespread of a tumour from its origin), the inhibition of invasion (thespread of tumour cells into neighbouring normal structures), or thepromotion of apoptosis (programmed cell death).

[0353] The present invention also provides active compounds which areuseful in the treatment of inflammatory conditions. For example, suchcompounds have growth down-regulatory effects on splenocytes. Examplesof inflammaotry conditions include, but are not limited to, rheumatoidarthritis, rheumatic fever, osteoarthritis, inflammatory bowel disease,psoriasis, and bronchial asthma.

[0354] The invention further provides active compounds for use in amethod of treatment of the human or animal body by therapy. Such amethod may comprise administering to such a subject atherapeutically-effective amount of an active compound, preferably inthe form of a pharmaceutical composition.

[0355] The term “treatment,” as used herein in the context of treating acondition, pertains generally to treatment and therapy, whether of ahuman or an animal (e.g., in veterinary applications), in which somedesired therapeutic effect is achieved, for example, the inhibition ofthe progress of the condition, and includes a reduction in the rate ofprogress, a halt in the rate of progress, amelioration of the condition,and cure of the condition. Treatment as a prophylactic measure is alsoincluded.

[0356] The term “therapeutically-effective amount,” as used herein,pertains to that amount of an active compound, or a material,composition or dosage from comprising an active compound, which iseffective for producing some desired therapeutic effect, commensuratewith a reasonable benefit/risk ratio.

[0357] The term “treatment” includes combination treatments andtherapies, in which two or more treatments or therapies are combined,for example, sequentially or simultaneously. Examples of treatments andtherapies include, but are not limited to, chemotherapy (theadministration of active agents, including, e.g., drugs, antibodies(e.g., as in immunotherapy), prodrugs (e.g., as in photodynamic therapy,GDEPT, ADEPT, etc.); surgery; radiation therapy; and gene therapy.

[0358] The invention further provides the use of an active compound forthe manufacture of a medicament, for example, for the treatment of aproliferative condition or an inflammatory condition, as discussedabove.

[0359] The invention further provides a method for regulating (e.g.,inhibiting) cell proliferation, comprising said cell with an effectiveamount of an active compound whether in vitro or in vivo.

[0360] Another aspect of the present invention pertains to methods oftreating a proliferative condition in a subject comprising administeringto said subject a therapeutically-effective amount of an activecompound, preferably in the form of a pharmaceutical composition.

[0361] Active compounds may also be used, as described above, incombination therapies, that is, in conjunction with other agents, forexample, cytotoxic agents.

[0362] Active compounds may also be used as part of an in vitro assay,for example, in order to determine whether a candidate host is likely tobenefit from treatment with the compound in question.

[0363] Active compounds may also be used as a standard, for example, inan assay, in order to identify other active compounds, otherantiproliferative agents, other antiinflammatory agents, etc.

[0364] Routes of Administration

[0365] The active compound or pharmaceutical composition comprising theactive compound may be administered to a subject by any convenient routeof administration, whether systemically/peripherally or topically (i.e.,at the site of desired action).

[0366] Routes of administration include, but are not limited to, oral(e.g, by ingestion); buccal; sublingual; transdermal (including, e.g.,by a patch, plaster, etc.); transmucosal (including, e.g., by a patch,plaster, etc.); intranasal (e.g., by nasal spray); ocular (e.g., byeyedrops); pulmonary (e.g., by inhalation or insufflation therapy using,e.g., via an aerosol, e.g., through the mouth or nose); rectal (e.g., bysuppository or enema); vaginal (e.g., by pessary); parenteral, forexample, by injection, including subcutaneous, intradermal,intramuscular, intravenous, intraarterial, intracardiac, intrathecal,intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal,intratracheal, subcuticular, intraarticular, subarachnoid, andintrasternal; by implant of a depot or reservoir, for example,subcutaneously or intramuscularly.

[0367] The Subject

[0368] The subject may be a prokaryote (e.g., bacteria) or a eukaryote(e.g., protoctista, fungi, plants, animals).

[0369] The subject may be a protoctista, an alga, or a protozoan.

[0370] The subject may be a plant, an angiosperm, a dicotyledon, amonocotyledon, a gymnosperm, a conifer, a ginkgo, a cycad, a fern, ahorsetail, a clubmoss, a liverwort, or a moss.

[0371] The subject may be an animal.

[0372] The subject may be a chordate, an invertebrate, an echinoderm(e.g., starfish, sea urchins, brittlestars), an arthropod, an annelid(segmented worms) (e.g., earthworms, lugworms, leeches), a mollusk(cephalopods (e.g., squids, octopi), pelecypods (e.g., oysters, mussels,clams), gastropods (e.g., snails, slugs)), a nematode (round worms), aplatyhelminthes (flatworms) (e.g., planarians, flukes, tapeworms), acnidaria (e.g., jelly fish, sea anemones, corals), or a porifera (e.g.,sponges).

[0373] The subject may be an arthropod, an insect (e.g., beetles,butterflies, moths), a chilopoda (centipedes), a diplopoda (millipedes),a crustacean (e.g., shrimps, crabs, lobsters), or an arachnid (e.g.,spiders, scorpions, mites).

[0374] The subject may be a chordate, a vertebrate, a mammal, a bird, areptile (e.g., snakes, lizards, crocodiles), an amphibian (e.g., frogs,toads), a bony fish (e.g., salmon, plaice, eel, lungfish), acartilaginous fish (e.g., sharks, rays), or a jawless fish (e.g.,lampreys, hagfish).

[0375] The subject may be a mammal, a placental mammal, a marsupial(e.g., kangaroo, wombat), a monotreme (e.g., duckbilled platypus), arodent (e.g., a guinea pig, a hamster, a rat, a mouse), murine (e.g., amouse), a lagomorph (e.g., a rabbit), avian (e.g., a bird), canine(e.g., a dog), feline (e.g., a cat), equine (e.g., a horse), porcine(e.g., a pig), ovine (e.g., a sheep), bovine (e.g., a cow), a primate,simian (e.g., a monkey or ape), a monkey (e.g., marmoset, baboon), anape (e.g., gorilla, chimpanzee, orangutang, gibbon), or a human.

[0376] Furthermore, the subject may be any of its forms of development,for example, a spore, a seed, an egg, a larva, a pupa, or a foetus.

[0377] In one preferred embodiment, the subject is a human.

[0378] Formulations

[0379] While it is possible for the active ingredient to be administeredalone, it is preferable to present it as a pharmaceutical composition(e.g., formulation) comprising at least one active ingredient, asdefined above, together with one or more pharmaceutically acceptablecarriers, excipients, buffers, adjuvants, stabilisers, or othermaterials well known to those skilled in the art and optionally othertherapeutic agents.

[0380] Thus, the present invention further provides pharmaceuticalcompositions, as defined above, and methods of making a pharmaceuticalcomposition comprising admixing at least one active ingredient, asdefined above, together with one or more pharmaceutically acceptablecarriers, excipients, buffers, adjuvants, stabilisers, or othermaterials, as described herein.

[0381] The term “pharmaceutically acceptable” as used herein pertains tocompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgement, suitable for use in contactwith the tissues of a subject (e.g., human) without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio. Each carrier,excipient, etc. must also be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation.

[0382] The formulations may conveniently be presented in unit dosageform and may be prepared by any methods well known in the art ofpharmacy. Such methods include the step of bringing into association theactive ingredient with the carrier which constitutes one or moreaccessory ingredients. In general, the formulations are prepared byuniformly and intimately bringing into association the active ingredientwith liquid carriers or finely divided solid carriers or both, and thenif necessary shaping the product.

[0383] Formulations may be in the form of liquids, solutions,suspensions, emulsions, tablets, losenges, granules, powders, capsules,cachets, pills, ampoules, suppositories, pessaries, ointments, gels,pastes, creams, sprays, foams, lotions, oils, boluses, electuaries, oraerosols.

[0384] Formulations suitable for oral administration (e.g., byingestion) may be presented as discrete units such as capsules, cachetsor tablets, each containing a predetermined amount of the activeingredient; as a powder or granules; as a solution or suspension in anaqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion ora water-in-oil liquid emulsion; as a bolus; as an electuary; or as apaste.

[0385] A tablet may be made by compression or moulding, optionally withone or more accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder (e.g., povidone, gelatin, hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (e.g., sodiumstarch glycolate, cross-linked povidone, cross-linked sodiumcarboxymethyl cellulose), surface-active or dispersing agent. Mouldedtablets may be made by moulding in a suitable machine a mixture of thepowdered compound moistened with an inert liquid diluent. The tabletsmay optionally be coated or scored and may be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile. Tablets may optionally beprovided with an enteric coating, to provide release in parts of the gutother than the stomach.

[0386] Formulations suitable for topical administration (e.g.,transdermal, intranasal, ocular, buccal, and sublingual) may beformulated as an ointment, cream, suspension, lotion, powder, solution,paste, gel, spray, aerosol, or oil. Alternatively, a formulation maycomprise a patch or a dressing such as a bandage or adhesive plasterimpregnated with active ingredients and optionally one or moreexcipients or diluents.

[0387] Formulations suitable for topical administration in the mouthinclude losenges comprising the active ingredient in a flavored basis,usually sucrose and acacia or tragacanth; pastilles comprising theactive ingredient in an inert basis such as gelatin and glycerin, orsucrose and acacia; and mouthwashes comprising the active ingredient ina suitable liquid carrier.

[0388] Formulations suitable for topical administration to the eye alsoinclude eye drops wherein the active ingredient is dissolved orsuspended in a suitable carrier, especially an aqueous solvent for theactive ingredient.

[0389] Formulations suitable for nasal administration, wherein thecarrier is a solid, include a coarse powder having a particle size, forexample, in the range of about 20 to about 500 microns which isadministered in the manner in which snuff is taken, i.e., by rapidinhalation through the nasal passage from a container of the powder heldclose up to the nose. Suitable formulations wherein the carrier is aliquid for administration as, for example, nasal spray, nasal drops, orby aerosol administration by nebuliser, include aqueous or oilysolutions of the active ingredient.

[0390] Formulations suitable for topical administration via the skininclude ointments, creams, and emulsions. When formulated in anointment, the active ingredient may optionally be employed with either aparaffinic or a water-miscible ointment base. Alternatively, the activeingredients may be formulated in a cream with an oil-in-water creambase. If desired, the aqueous phase of the cream base may include, forexample, at least about 30% w/w of a polyhydric alcohol, i.e., analcohol having two or more hydroxyl groups such as propylene glycol,butane-1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycoland mixtures thereof. The topical formulations may desirably include acompound which enhances absorption or penetration of the activeingredient through the skin or other affected areas. Examples of suchdermal penetration enhancers include dimethylsulfoxide and relatedanalogues.

[0391] When formulated as a topical emulsion, the oily phase mayoptionally comprise merely an emulsifier (otherwise known as anemulgent), or it may comprises a mixture of at least one emulsifier witha fat or an oil or with both a fat and an oil. Preferably, a hydrophilicemulsifier is included together with a lipophilic emulsifier which actsas a stabiliser. It is also preferred to include both an oil and a fat.Together, the emulsifier(s) with or without stabiliser(s) make up theso-called emulsifying wax, and the wax together with the oil and/or fatmake up the so-called emulsifying ointment base which forms the oilydispersed phase of the cream formulations.

[0392] Suitable emulgents and emulsion stabilisers include Tween 60,Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearateand sodium lauryl sulfate. The choice of suitable oils or fats for theformulation is based on achieving the desired cosmetic properties, sincethe solubility of the active compound in most oils likely to be used inpharmaceutical emulsion formulations may be very low. Thus the creamshould preferably be a non-greasy, non-staining and washable productwith suitable consistency to avoid leakage from tubes or othercontainers. Straight or branched chain, mono- or dibasic alkyl esterssuch as di-isoadipate, isocetyl stearate, propylene glycol diester ofcoconut fatty acids, isopropyl myristate, decyl oleate, isopropylpalmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branchedchain esters known as Crodamol CAP may be used, the last three beingpreferred esters. These may be used alone or in combination depending onthe properties required. Alternatively, high melting point lipids suchas white soft paraffin and/or liquid paraffin or other mineral oils canbe used.

[0393] Formulations suitable for rectal administration may be presentedas a suppository with a suitable base comprising, for example, cocoabutter or a salicylate.

[0394] Formulations suitable for vaginal administration may-be presentedas pessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient, such carriers as areknown in the art to be appropriate.

[0395] Formulations suitable for parenteral administration (e.g., byinjection, including cutaneous, subcutaneous, intramuscular, intravenousand intradermal), include aqueous and non-aqueous isotonic,pyrogen-free, sterile injection solutions which may containanti-oxidants, buffers, preservatives, stabilisers, bacteriostats andsolutes which render the formulation isotonic with the blood of theintended recipient; and aqueous and non-aqueous sterile suspensionswhich may include suspending agents and thickening agents, and liposomesor other microparticulate systems which are designed to target thecompound to blood components or one or more organs. Examples of suitableisotonic vehicles for use in such formulations include Sodium ChlorideInjection, Ringer's Solution, or Lactated Ringers Injection. Typically,the concentration of the active ingredient in the solution is from about1 ng/ml to about 10 μg/ml, for example from about 10 ng/ml to about 1μg/ml. The formulations may be presented in unit-dose or multi-dosesealed containers, for example, ampoules and vials, and may be stored ina freese-dried (lyophilised) condition requiring only the addition ofthe sterile liquid carrier, for example water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules, and tablets.Formulations may be in the form of liposomes or other microparticulatesystems which are designed to target the active compound to bloodcomponents or one or more organs.

[0396] Dosage

[0397] It will be appreciated that appropriate dosages of the activecompounds, and compositions comprising the active compounds, can varyfrom patient to patient. Determining the optimal dosage will generallyinvolve the balancing of the level of therapeutic benefit against anyrisk or deleterious side effects of the treatments of the presentinvention. The selected dosage level will depend on a variety of factorsincluding, but not limited to, the activity of the particular compound,the route of administration, the time of administration, the rate ofexcretion of the compound, the duration of the treatment, other drugs,compounds, and/or materials used in combination, and the age, sex,weight, condition, general health, and prior medical history of thepatient. The amount of compound and route of administration willultimately be at the discretion of the physician, although generally thedosage will be to achieve local concentrations at the site of actionwhich achieve the desired effect.

[0398] Administration in vivo can be effected in one dose, continuouslyor intermittently throughout the course of treatment. Methods ofdetermining the most effective means and dosage of administration arewell known to those of skill in the art and will vary with theformulation used for therapy, the purpose of the therapy, the targetcell being treated, and the subject being treated. Single or multipleadministrations can be carried out with the dose level and pattern beingselected by the treating physician.

[0399] In general, a suitable dose of the active compound is in therange of about 0.1 to about 250 mg per kilogram body weight of thesubject per day. Where the active ingredient is a salt, an ester,prodrug, or the like, the amount administered is calculated on the basisthe parent compound and so the actual weight to be used is increasedproportionately.

[0400] Prodrugs

[0401] Compounds of the present invention may be prodrugs for potentantiproliferative agents. Compounds which exhibit low or moderateintrinsic activity may act as prodrugs, and be metabolically activated(e.g., in vivo) to generate more potent compounds. This is especiallyuseful in cancer therapy where metabolic activation can be achieved byan enzyme that is expressed in tumours.

[0402] For example, the cytochrome P-450 enzyme CYP1B1 has been shown tobe specifically expressed in tumour cells, but is not found in thecorresponding normal tissues. This enzyme is found to be expressed in avariety of tumours, such as brain, breast, colon, stomach, ovarian andprostate cancers (see, e.g., Murray et al, 1997; Melvin et al., 1997).Prodrugs, acting as a substrate, may be metabolised by CYP1B1 through anaromatic hydroxylation reaction to generate a potent anticancer agent.

[0403] For example, as illustrated below, a prodrug, with low intrinsicactivity (e.g., IC50 of 0.69 μM in breast cancer MCF-7 cells)(E)-1-(4-methoxyphenyl)-3-(3,5-dimethoxyphenyl)prop-1-en-3-one, isconverted to the hydroxylated metabolite,(E)-1-(3-Hydroxy-4-methoxyphenyl)-3-(3,5-dimethoxyphenyl)prop-1-en-3-one, which has substantially potency (e.g., Iq50 of 0.00065μM in the same cell line).

[0404] Thus, those compounds of the present invention where R^(A3) is —Hmay be prodrugs, to be activated by CYP1B1 enzyme, to yield thecorresponding drug where R^(A3) is —OH.

[0405] In such cases, the prodrug is useful as an antiproliferativeagent with low intrinsic toxicity, for treatment of proliferativeconditions characterised by cells which express the CYP1B1 enzyme.

[0406] Additionally, the prodrug is useful as a selectiveantiproliferative agent with low intrinsic toxicity, for treatment ofproliferative conditions characterised by cells which express the CYP1B1enzyme, where the corresponding normal cells do not express the CYP1B1enzyme.

[0407] Furthermore, prodrugs with low intrinsic cytotoxicity, which areonly activated upon entering cells (e.g., tumour cells) containing theCYP1B1 enzyme, are not only useful for treating cancer, but also as aprophylactic, in cancer prevention (i.e., as a cancer preventativeagent).

[0408] A method for detecting and/or demonstrating the conversion of acandidate prodrug to the corresponding drug is described next: Amicrosomal preparation of human tumour tissue expressing the CYP1B1enzyme is prepared essentially as described by the method of Barrie etat., 1989. The experiment is carried out at 37° C., under yellow light.An array of 1.5 ml centrifuge tubes are set up in a water bath shakerunder aerobic conditions. To each tube is then added 500 μl of pH 7.6buffer (0.1 M NaK₂PO₄), followed by NADPH (5 μl of a 25 mM stocksolution). The microsomal preparation (80 μl) is then added and thetubes pre-incubated for 5 min at 37° C. The prodrug is then added (10 μlof a 5 mM stock solution) and the preparation incubated for 1 h at 37°C. After 1 h the tubes are transferred to an ice/water cooling bath (0°C.). The tubes are then centrifuged at 15,000 rpm for 30 min. A sampleof the supernatant (100 μl) is then taken and analysed by HPLC. HPLCconditions: Spherisorb C18 (25 cm×4.6 mm id), used without guard column.Flow rate 1 ml/min. Eluent 75% 0.1 M KH₂PO₄ and 25% acetonitrile. Thehydroxylated drug is detected by HPLC, and confirmed by comparison withthe authentic hydroxylated synthetic compound.

[0409] Diagnosis and Assays

[0410] In many cases, hydroxylated compounds, where R^(A3) is —OH,exhibit much greater fluorescence than the correspondingnon-hydroxylated compound, where R^(A3) is —H. This property may beexploited in diagnosis, for example, of cancer, by detecting and/ormeasuring the formation of the hydroxylated metabolite via tumour cellsexpressing the CYP1B1 enzyme.

[0411] Thus, one aspect of the present invention pertains to a method ofdiagnosis of a subject for the presence of cells (e.g., tumour cells)expressing the CYP1B1 enzyme, comprising:

[0412] (a) administering to the patient a non-hydroxylated prodrug asdescribed herein, wherein R^(A3) is —H;

[0413] (b) determining the amount of the corresponding hydroxylatedmetabolite, wherein R^(A 3) is —OH which is subsequently produced; and,

[0414] (c) correlating the amount with the presence or absence of thecells in the patient.

[0415] Another aspect of the present invention pertains to activecompounds, wherein R^(A3) is —H, for use in a method of diagnosis of thehuman or animal body. In one embodiment, the diagnosis is for thepresence of cells (e.g., tumour cells) expressing the CYP1B1 enzyme.

[0416] Another aspect of the present invention pertains to use of activecompounds, wherein R^(A3) is —H, for the manufacture of a composition,for example, for the diagnosis of the presence of cells (e.g., tumourcells) expressing the CYP1B1 enzyme, a proliferative condition, aninflammatory condition, etc., as discussed above.

[0417] Kits

[0418] One aspect of the invention pertains to a kit comprising (a) theactive ingredient, preferably provided in a suitable container and/orwith suitable packaging; and (b) instructions for use, for example,written instructions on how to administer the active compound, how toperform a diagnosis using the active compound, etc.

[0419] The written instructions may also include a list of indicationsfor which the active ingredient is a suitable treatment.

EXAMPLES

[0420] The following are examples are provided solely to illustrate thepresent invention and are not intended to limit the scope of theinvention, as described herein.

[0421] Analytical Methods

[0422] The ¹H— and ¹³C-NMR spectra were recorded on a 250 MHzsuper-conducting Bruker AC250 Spectrometer. Infrared spectra wererecorded in potassium bromide on a Shimadzu FTIR-8300 Spectrophotometer.The mass spectra were recorded on a VG 70 SEQ Spectrometer. Meltingpoints were determined on an Electrothermal melting point apparatus andwere uncorrected. Thin layer chromatography was performed on silica gelsheets (Merck TLC Aluminium sheet-Silica Gel 60F) and was monitored withUV light. Column chromatography was performed using Silica gel 60(220440 mesh).

Example 1(E)-1-(3,4-Methylenedioxyphenyl)-3-(3,4,5-trimethoxyphenyl)prop-1-en-3-one(DMU-135)

[0423]

[0424] To a stirred solution of piperonal (1.13 g, 7.5 mmol) and3,4,5-trimethoxyacetophenone (1.58 g, 7.5 mmol) in methanol (15 ml) wasadded aqueous NaOH (6 ml, 50% w/v) and the mixture stirred for 18 h. Theresultant solid was collected by filtration and recrystallised frommethanol (100 ml) as pale yellow crystals (1.98 g, 77%): mp 135° C.; ¹HNMR δ (CDCl₃) 3.92 (3H, s, OCH₃), 3.93 (6H, s, OCH₃), 6.01 (2H, s, CH₂),6.83 (1H, d, J=8.0 Hz, H-5), 7.11 (1H, dd, J=1.6, 8.0 Hz, H-6), 7.15(1H, d, J=1.6 Hz, H-2), 7.25 (2H, s, H-2′,6′), 7.30 (1H, d, J=15.5 Hz,CH), 7.72 (1H, d, J=15.5 Hz, CHCO); ¹³C NMR 56.42, 60.99, 101.69,106.06, 106.68, 108.72, 119.77, 125.26, 133.72, 142.43, 144.60, 148.44,149.96, 153.17, 189.07; MS (rel intensity) m/z 343 ([M+H]⁺, 100%); Anal.Calcd (C₁₉H₁₈O₆): C, 66.66; H, 5.30. Found C, 66.43; H, 5.45.

Example 2(E)-1-(3,4-methylenedioxyphenyl)-3-(3,5-dimethoxyphenyl)prop-1-en-3-one(DMU403)

[0425]

[0426] To a stirred solution of Piperonal (1.00 g, 6.67 mmol) and3,5-dimethoxyacetophenone (1.20 g, 6.67 mmol) in methanol (30 ml) wasadded a 50% w/v solution of aqueous NaOH (8 ml, 15 eq). A colourlesssolution had formed initially following the dissolution of the twostarting materials in the solvent, but upon addition of NaOH, thesolution became a yellow, creamy suspension. A pale yellow precipitatewas noticed approximately 2 or 3 mins after adding the aqueous NaOH. Afurther 3 ml (−5 eq) of base was added, after 5 h, in order to drive thereaction to completion. The mixture was stirred for a total of 6 h atroom temperature. The precipitate was collected via vacuum filtrationand the filtrate discarded. The solid was purified by recrystallisationfrom hot methanol and hot filtration. The product was collected andallowed to be dried under vacuum to afford the title compound as a paleyellow solid (0.53 g, 25%). ¹H-NMR (CDCl₃) δ 7.25 (1H,d,CH(9)),7.30(2H,d,CH(6&8)), 6.85 (1H,d,CH(3)), 6.65 (1H,t,CH(4)), 6.00(2H,s,CH₂(1)), 3.85 (6H,s,OCH₃(5&7)); ¹³C-NMR (CDCl₃) δ 189.958 (C═O),160.870, 149.928, 148.401, 144.756, 140.403, 129.325, 125.221, 120.112,108.650, 106.659, 106.253, 104.879, 101.621, 55.608 (OCH₃); InfraredSpectrum V_(max) (KBr)/cm⁻¹ 1668.3 (C═O); Mass Spectrum (FAB) m/e 313(m+1); Elemental Analysis: Molecular Compound C₁₈H₁₆O₅, calculatedC=69.23, H=5.19 and found C=69.15, H=5.18.

Example 3(E)-1-(3,4-methylenedioxyphenyl)-3-(2,4-dimethoxyphenyl)prop-1-en-3-one(DMU407)

[0427]

[0428] To a stirred solution of piperonal (1 g, 6.7 mmol) and2,4-dimethoxyacetophenone (1.21 g, 6.7 mmol) in methanol (30 ml) wasadded 20 equivalents of aqueous sodium hydroxide (˜11 ml, 50% w/v) andthe mixture stirred for 2 h. The resultant solid was collected byfiltration and then recrystallised from methanol as pale yellow crystals(0.86 g, 41%): mp 137° C.; ¹H NMR δ (CDCl₃) 3.9 (3H, s, OCH₃), 3.92 (3H,s, OCH₃), 6.01 (2H, s, CH₂O), 6.50 (2H, dd, ArH), 6.80 (1H, d, ArH),7.00 (2H, m, ArH), 7.25 (1H, d, J=15.7 Hz, ArH), 7.59 (1H, d, J=15.7 Hz,ArH), 7.70 (1H, d, ArH); ¹³C NMR δ 101.45, 122.35, 129.93, 148.23,149.37, 160.29, 164.05, 190.34; Infra red vMAX (KBr)/cm⁻¹ 1659.6 (C═O);MS (rel intensity) m/z 313 ([M+H]⁺, 37%); Anal. Calcd (CO₈H₁₆O₅): C,69.23; H, 5.13. Found C, 68.97; H, 5.26.

Example 4(E)-1-(3,4-methylenedioxyphenyl)-3-(2,3,4-trimethoxyphenyl)prop-1-en-3-one(DMU419)

[0429]

[0430] To a stirred solution of piperonal (0.714 g, 4.8 mmol) and2,3,4-trimethoxyacetophenone (1.0 g, 4.8 mmol) in methanol (30 ml) wasadded a 50% w/v solution of aqueous sodium hydroxide (NaOH) (5.70 ml, 15eq). A clear, pale yellow coloured solution was formed initially andprogressed to a darker yellow/green colour that was less clear.Eventually a pale yellow precipitate was formed on the sides, togetherwith pale yellow oil droplets at the base of the flask.

[0431] More aqueous NaOH solution (1.90 ml, 5 eq) was added. Thisresulted in the immediate precipitation of more pale yellow solid andcomplete disappearance of the oil droplets. The reaction was allowed toreach completion and the resultant solid collected by vacuum filtration,and washed with a small quantity of cold methanol. The solid wasrecrystallised from hot methanol and dried under vacuum to afford thetitle compound as pale yellow needle shaped crystals (1.004 g, 61%).¹H-NMR (CDCL₃, 250 MHz) δ 7.55 (3)(1H,d), 7.43(1)(1H,d), 7.28 (4)(1H,d),7.08 (5)(1H,m), 7.03 (6)(1H,m), 6.78 (7)(1H,d), 6.72 (2)(1H,d), 6.00(CH₂)(2H,s), 3.92 (OCH₃)(3H,s), 3.91 (OCH₃)(3H,s), 3.90 (OCH₃)(3H,s);¹³C-NMR (CDCl₃, 250 MHz) δ 190.697 (C═O); Infrared Spectrum ν_(max)(KBr)/cm⁻¹ 1653.8 (C═O); Mass Spectrum (FAB) m/e 343 (M+1); ElementalAnal. Calcd for C₁₉H₁₈O₆: C, 66.66; H, 5.30. Found: C, 66.56; H, 5.43.

Example 5(E)-1-(3,4-methylenedioxyphenyl)-3-(2,5-dimethoxyphenyl)prop-1-en-3-one(DMU423)

[0432]

[0433] To a stirred solution of piperonal (1 g, 6.7 mmol) and2,5-dimethoxyacetophenone (1.21 g, 6.7 mmol) in methanol (30 ml) wasadded 15 equivalents of aqueous sodium hydroxide (−8 ml, 50% w/v) andthe mixture stirred for 2 h. The resultant solid was collected byfiltration and then recrystallised from methanol as fine, needle-likepale yellow crystals (1.70 g, 81%): mp 101° C.; ¹H NMR δ (CDCl₃) 3.78(3H, s, OCH₃), 3.85 (3H, s, OCH₃), 6.00 (2H, s, CH₂O), 6.83 (1H, d,ArH), 6.90 (1H, d, ArH), 7.00 (3H, m, ArH), 7.15 (1H, d, ArH), 7.23 (1H,d, J=15.7 Hz, ArH), 7.52 (1H, d, J=15.8 Hz, ArH); Infra red ν_(max)(KBr)/cm⁻¹ 1652.9 (C═O); MS (rel intensity) m/z 313 ([M+H]⁺, 100%);Anal.

[0434] Calcd (C₁₈H₁₆O₅): C, 69.23; H, 5.13. Found C, 69.25; H, 4.88.

Example 6(E)-1-(3,4-methylenedioxyphenyl)-3-(4-hydroxy-3,5-dimethoxyphenyl)prop-1-en-3-one(DMU-452)

[0435]

[0436] The title compound was obtained by a method analogous to that ofExample 1, and using 4-hydroxy-3,5-dimethoxyacetophenone instead of3,4,5-trimethoxyacetophenone.

Example 7 (E)-1-(5-hydroxy-3,4-methylenedioxyphenyl)-3-(3,4,5-trimethoxyphenyl)prop-1-en-3-one (DMU458)

[0437]

[0438] The title compound was obtained by a method analogous to that ofExample 1, and using 5-hydroxy-3,4-methylenedioxybenzaldehyde instead ofpiperonal.

[0439] Biological Activity

[0440] TCDD-Induced MCF-7 Cell Line Versus MCF-7 Cell Line CytotoxicityAssay

[0441] CYP1A1 and CYP1B1 enzyme activity is induced by TCDD(tetrachlorodibenzodioxin (Dioxin)) in breast tumour MCF-7 cells (see,e.g., Sutter et al., 1994). CYP1B1 is expressed in a variety of humantumours, and can be inducible by TCDD in numerous cell types includingbreast, liver, lung, and kidney (see, e.g., Murray et al., 1997). CYP1B1is known to catalyse estradiol 4-hydroxylation metabolism. In untreatedculture, the constitutive rate of estradiol E₂ metabolism in MCF-7 cellsis minimal. However, treatment with TCDD causes a marked increase in therate of E₂ metabolism (see, e.g., Spink et al., 1994). Thus

[0442] MCF-7 cells in culture that are non-induced are metabolicallyanalogous to normal cells that do not express CYP1B1, whilstTCDD-induced MCF-7 cells express the CYP1B1 enzyme as is present infresh human tumours. Therefore the cytotoxicity of compounds innon-induced MCF-7 cells correlates to the cytotoxicity of compoundsagainst normal cells, whilst the cytotoxicity of compounds againstTCDD-induced MCF-7 cells correlates to the cytotoxicity of compoundsagainst real tumours that express CYP1B1. In this assay, a tumourselectivity factor greater than 1 (and preferably greater than 1.5) ishighly significant and demonstrates that the compound has tumourselective cytotoxic activity.

[0443] The non-induced MCF-7 cell line is analogous enzymatically tonormal cells that do not express catalytically active CYP1 familyenzymes. The cytotoxicity of compounds in non-induced MCF-7 cellscorrelates to the cytotoxicity of compounds against normal cells, whilstthe cytotoxicity of compounds against TCDD-induced MCF-7 cellscorrelates to the cytotoxicity of compounds against real tumours thatexpress CYP1B1.

[0444] Cells were maintained in RPMI 1640 with Glutamax 1 (LifeTechnologies) with 10% (v/v) heat inactivated foetal calf serum(Hybrimax. Sigma), at 37° C., 5% CO₂/95% air with 100% humidity andpassaged using trypsin/EDTA. 1×10³ cells were plated out in 100 μlmedium per well of 96-well flat-bottomed plates (Fisher). After 4 hoursto allow adherance, 100 μl of medium containing TCDD (British GreyhoundChromatography; 10 μM stock in DMSO (dimethylsulfoxide)) or medium with0.2% (v/v) DMSO as control was added to each well to give a finalconcentration of 10 nM TCDD, 0.1% (v/v) DMSO, for 24 hours to allowmaximal CYP expression. The medium was then carefully aspirated and 100μl fresh medium added. Within 30 minutes test compound was added inquadruplicate in 100 μl medium (with or without inhibitors) at doublethe final concentration from 100 mM stock in DMSO to give a finalconcentration of not more than 0.1% (v/v) DMSO, or DMSO solvent alone at0.1% (v/v) as control. The cells were then allowed to grow on for 96hours to give 80-90% confluence in the control wells. 50 μl MTT (Thiazolblue, Sigma) at 2 mg/ml in Dulbecco's phosphate buffered saline-A, wasthen added to each well for 3 hours: All medium was aspirated, then theformazan product generated by viable cells was solubilized with 150 piDMSO. Plates were vortexed and the absorbance at 540 nm determined usinga plate reader. Results were expressed as the percentage of 100%(control) proliferation and the IC50 calculated using the line of bestfit for a sigmoidal dose response curve with variable slope usingGraphpad Prizm software. All determinations were carried out in at leasttriplicate.

[0445] The selectivity differential factor (TSDF) is calculated bydividing the IC50 obtained from the MCF-10A data by the IC50 obtainedfrom the MDA468 data.

[0446] A selectivity factor greater than 1 (and preferably greater than1.5) is highly significant and demonstrates that the compound has tumourselective cytotoxic activity.

[0447] The results of this assay are summarised in the table below.Compound DMU-135 is 65-fold more toxic to “tumour” cells than to“normal” cells. Compounds DMU411, DMU-416, DMU-160, and DMU-104 showedlittle or no selectivity. Compound DMU-103 is 2-fold more toxic tonormal cells than to cancer cells. TABLE 1 Cytotoxicity TCDD- inducedTumor MCF-7 MCF-7 Selectivity Cells Cells Differential Compound IC50(μM) IC50 (μM) Factor DMU-135 0.92 0.014 65 DMU-403 3.5 0.57 6 DMU-407 72.7 3 DMU-419 2.8 0.84 3 DMU-423 3.5 0.26 13 DMU-411 4.2 4.2 1.0 DMU-4168.9 8.9 1.0 DMU-160 14.0 14.0 1.0 DMU-103 0.04 0.08 0.5 DMU-104 3.0 3.01.0

[0448]FIG. 1 is a graph of cell survivial (%) versus concentration (μM)of compound DMU-135, for (A) the TCDD-induced MCF-7 cell line (▪) and(B) the MCF-7 cell line (▾).

[0449] This graph shows that compound DMU-135 has an IC50 of 0.92 μM inun-induced MCF-7 cells, but has an IC500.014 μM in TCDD-induced MCF-7cells. This illustrates a surprising and unexpected 65-fold increase inthe cytotoxic activity of DMU-135 by the induction of CYP1B1.Consequently, DMU-135 has a large therapeutic window; is active at muchlower doses; and will specifically target the tumour cells that expressCYP1B1, whilst normal cells will be preferentially spared.

[0450] MDA468 Tumour Cell Line Versus MCF-10A Normal Cell Line Assay

[0451] This cell culture based assay is performed using the two celllines MDA-468 and MCF-10A. The MDA-468 cell line is an advanced breastcancer cell line, whilst the MCF-10A cell line is a normal breast cellline.

[0452] This assay was performed using the two cell lines MDA468 andMCF-10A according to the procedure described above for the MCF-7 assay,but without the addition of TCDD.

[0453] The tumour selectivity differential differential factor(TSDF) iscalculated by dividing the IC50 obtained from the MCF-10A data by theIC50 obtained from the MDA-468 data. In this assay, a tumour selectivityfactor greater than 1 (and preferably greater than 1.5) is highlysignificant and demonstrates that the compound has tumour selectivecytotoxic activity.

[0454] The results of this assay on Compound DMU-135, together with theclinically used anticancer agents tamoxifen, methotrexate, anddoxorubicin (adriamycin) for comparison, are summarised in the tablebelow. Compound DMU-135 is 120-fold more toxic to cancer cells than tonormal cells. In contrast, the clinically used anticancer agentDoxorubicin is actually found to be 10-fold more toxic to normal cellsthan to cancer cells. TABLE 2 Cytotoxicity Tumor MDA-468 MCF-10ASelectivity (Breast Tumor) (Normal Breast) Differential Compound IC50(uM) IC50 (uM) Factor DMU-135 0.02 2.3 120 Tamoxifen 4.0 6.3 1.6Methotrexate 0.04 0.06 1.5 Doxorubicin 0.003 0.0003 0.1

[0455]FIG. 2 is a graph of cell survivial (%) versus concentration (μM)of compound DMU-135, for (A) the normal breast cell line MCF-10A (∘),and (B) the advanced breast cancer cell line MDA-468 ().

[0456] This graph shows that compound DMU-135 shows a low toxicity IC50value of 2.3 μM against the normal cell line, but a highly potent IC50value of 0.02 μM against the advanced tumour cell line. This illustratesa surprising and unexpected 120-fold tumour selectivity in the cytotoxicactivity of DMU-135.

[0457] Splenocyte Anti-Proliferation Assay

[0458] The splenocyte anti-proliferation assay has been developed toidentify compounds that have useful anti-inflammatory properties for thetreatment of auto-inflammatory diseases such as rheumatoid arthritis.See, for example, Yamashita et al., 1994. This well known assay isdescribed in detail in, for example, Mosmann, 1983. In this assay,splenocyte proliferation is stimulated by the inflammatory responseinducer conconavilin A (Con A). Cell proliferation is monitored bydetecting radiation (counts per minute, cpm) from a radio label(tritiated thymidine) which is incorporated only into proliferatingcells.

[0459] For example, compounds may be assayed as a solution indimethylsulfoxide (DMSO) as solvent. A solvent control may also betested for comparison. Other controls may be used. Compounds thatexhibit anti-inflammatory effects at a concentration of less than 10 μMare considered to be useful therapeutic agents.

[0460] The compounds of the present invention also show growthdown-regulatory effects on splenocytes. Since splenocytes are involvedin inflammation, these compounds are also useful as anti-inflammatoryagents.

REFERENCES

[0461] A number of patents and publications are cited above in order tomore fully describe and disclose the invention and the state of the artto which the invention pertains. Full citations for these references areprovided below. Each of these references is incorporated herein byreference in its entirety into the present disclosure.

[0462] Barrie, S. E., et al., 1989, “Inhibition of17-hydroxylase/C17-C20 Lyase by Bifluranol and Its Analogues,” J.Steroid Biochem., Vol. 33, No. 6, pp. 1191-1195.

[0463] Berryman et al., 1995, published international (PCT) patentapplication number WO 95/05376, published 23 Feb. 1995.

[0464] Berryman et al., 1997, U.S. Pat. No. 5,691,373, granted 25 Nov.1997.

[0465] Carmichael, J., et al., 1987, “Evaluation of a Tetrazolium-basedSemiautomated Colorimetric Assay: Assessment of ChemosensitivityTesting,” Cancer Research, Vol. 47, p. 936-942.

[0466] Cushman et al., 1995, U.S. Pat. No. 5,430,062, granted 04 Jul.1995.

[0467] Ducki, S., et al., 1998, “Potent Antimitotic and Cell growthInhibitory Properties of Substituted Chalcones,” BioMed. Chem. Lett.,Vol. 8, pp. 1051-1056.

[0468] Eda Shoei et al., 1986, Japanese patent publication numberJP-61-076433A (application number JP-59-199262) published 18 Apr. 1986.

[0469] Hall et al., 1981, U.S. Pat. No. 4,279,930, granted 21 Jul. 1981.

[0470] Ikeda Shunichi et al., 1996, Japanese patent publication numberJP-08-188546A (application number JP-07-000002) published 23 Jul. 1996.

[0471] Kharazmi et al., 1999, published international (PCT) patentapplication number WO 99/00114, published 07 Jan. 1999.

[0472] Melvin et al., 1997, published international (PCT) patentapplication number WO 97/12246, published 03 Apr. 1997.

[0473] Mosmann, T., 1983, “Rapid Colorimetric Assay for Cellular Growthand Survival: Application to Proliferation and Cytotoxicity Assays,”Journal of Immunological Methods, Vol. 65, pp. 55-63.

[0474] Murray, G. I., et al., 1997, “Tumour-specific Expression ofCytochrome P450 CYP1B1,” Cancer Research, Vol. 57, pp. 3026-3031.

[0475] Pettit, G. R., et al., 1995, “Antineoplastic agents 322.Synthesis of Combretastatin A4 Prodrugs,” Anticancer Drug Design, Vol.10, pp. 299-309.

[0476] Potter et al., 1999, published international (PCT) patentapplication number WO 99/40056, published 12 Aug. 1999.

[0477] Potter et al., 2001a, U.S. Pat. No. 6,214,886, granted 10 Apr.2001.

[0478] Potter et al., 2001b, published international (PCT) patentapplication number WO 01/72680, published 04 Oct. 2001.

[0479] Spink, D. C., et al., 1994, “The Effects of2,3,7,8-Tetrachlorodibenzo-p-dioxin on Estrogen Metabolism in MCF-7Breast Cancer Cells: Evidence for Induction of a Novel 17β-Estradiol4-hydroxylase,” J. Steroid Biochem. Mol. Biol., Vol. 51, No. 5/6, pp.251-258.

[0480] Sutter, T. R., et al, 1994, “Complete cDNA sequence of a humandioxin-inducible mRNA identifies a new gene subfamily of cytochrome P450that maps onto chromosome 2,” J. Biol. Chem., Vol. 269, No. 18, pp.13092-13099.

[0481] Yamashita, D. S., et al, 1994, “Design, Synthesis and Evaluationof Dual Domain FKBP Ligands,” Bioorg. Med. Chem. Lett., Vol. 4, No. 2,pp.325-328.

1. Use of a compound for the manufacture of a medicament for use in thetreatment of a proliferative condition characterised by cells whichexpress CYP1B1, wherein the compound has the following formula:

wherein: each of R^(B2), R^(B3), R^(B4), and R^(B5) is independently —H,—OH, or —OMe; each of R¹ and R² is independently: —H, optionallysubstituted C₁₋₄alkyl, or optionally substituted C₅₋₂₀aryl; R^(A3) is—H, —OH, —OC(═O)R^(E), —OS(═O)₂OH, or —OP(═O)(OH)₂; R^(E) is: —H,optionally substituted C₁₋₆alkyl, optionally substitutedC₃₋₂₀heterocyclyl, or optionally substituted C₅₋₂₀aryl; or apharmaceutically acceptable salt, solvate, amide, ester, ether,chemically protected form, or prodrug thereof. 2-118. (Canceled).